JPH07501923A - small protein - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] small protein Background of the invention [Field of invention] This invention relates to the development of novel small binding proteins, especially micro The process involves protein mutagenesis, expression, affinity selection, and amplification. This research concerns the development of new binding proteins by recombination. This process In this process, genes encoding binding regions of small proteins are The gene obtained by serendipitous mutagenesis of the constant codon It's melting. The genetic elements can be found in viruses (particularly filamentous phages) or cells. Generate a resulting mixture of cell population expression displayed on the outer surface. affinity Selection to identify genomes of viruses or cells containing fused cells It's being used. Molten cells bind proteins to chromatographic targets Attach a sign to

[Description of related technology] A protein's amino acid sequence determines its three-dimensional (3D) structure. This is true determine the function of the protein. Some residues (residues, hereafter) of the polypeptide chain (same) plays an important role in determining the three-dimensional structure of proteins. follow Its binding ability is not a covalent bond but a close bond and specificity, which is Combine with the characteristics of the target molecule. "Protein engineering" is the order of protein sequences It is a technology to operate. That is, to change the properties of that bond. protein Although the factors that influence binding are known, novel surface designs are difficult. I know that there is. Quiocho and others (QUI087) are as follows Suggests. That is, current protein engineering methods cannot It is difficult to create proteins with the excellent binding properties of proteins. Dew.

However, there is a track record of some isolated successes. For example, filkins on et al. (NIL 84) reported that mutant THr3. --Bacil with Pro lus ste-arothermophilus tyrosine transfer RN A mutant of A synthetase has a 1°00-fold increase in affinity for ATP. It is reported that this is shown.

With the development of recombinant DNA technology, it is now possible to attach genetic codes to natural proteins and mutate them suddenly. Create mutant proteins by mutating the gene and expressing the mutated gene. Now you can. (Some methods of mutagenesis are known.

One is “Protein Surgery J (DILL87),” which involves It is the introduction of one or more targeted mutations. Perfect A unique polypeptide of the predicted sequence has been expressed and its binding properties evaluated. There is.

At the other extreme, relatively unspecified mutagenic agents such as radiation or various chemicals are used. It is a method that randomly generates mutants using a stimulant. InOther()l OcJ85 and Lehtnvanrs, EP Appln, 285.123 checking ...

Predetermined nucleic acids can be synthesized using mixtures of various groups in appropriate cycles of nucleic acid synthesis procedures. You can change leotide randomly.

(OLIP86 and 0LP87). The proportion of groups in the mixture is determined by the position of each codon. The frequency is determined according to the location. Each amino acid at that frequency (yo, degenerated DNA solids occurs in polypeptides expressed from groups.

(REUD88a, VER386a% VER586b). The difference that codes for DNA Please mention the difference in the abundance of amino acids that have changed (L).

Ferenci and co-workers identified the maltose transport protein E, Co11. He has published a series of papers on the chromatographic isolation of LamB. (FERE 82a, FERE82b, FERE83, FERE84, CL11N8 4,) iK I 8B? , and the above-mentioned paper) mutants are caused by unspecified chemical reactions. Produced spontaneously or induced by natural mutagens. The level of mutagenesis selected, defining a single point mutation or a single insertion of two residues. multiple mutations is a power that is neither explored nor discovered.

Variations in the degree of affinity for conventional LamB substrates maltose and starch are observed. On the other hand, the parent for a molecule as a target that is not bound by natural LamB Selection of compatibility 1) Also, multiple mutations are not explored or discovered. Mona power ＼tsuta.

Literature FERE84 is based on affinity chromatographic selection technology (other It can be applied to generate mutants similar to "key bacterial enzymes present in , and also selected mutations that relocate intracellular bacterial proteins to the cell surface. I think it can be done. However, Ferenci's mutation Body surface proteins may be exogenous or non-identical to bacterial surface proteins. It was a chimera in the combined area.

Ferenci also cloned structural genes and determined protein structures, active regions, and They argue that it is not necessary to know the position or sequence.

However, the method according to the invention specifically utilizes cloned structural genes. There are many things. sign to a defined potential binding protein on the outer surface of the chimera It is not possible to construct and express a gene marked with `` without cloning it.

Ferenci did not limit mutations to special loci. Replacement is a special part It is not limited by the desirability of specific amino acid types at positions, but rather by mutagenesis. Limited by the properties of matter. In this invention, the protein structure, Knowledge of the active site and sequence is used to make appropriate predictions. That knowledge will determine which residues Predicting which will most significantly affect binding activity without unduly destabilizing the protein. assistance and mutagenesis is focused on these sites. Ferenci is There is no suggestion that surface residues should be favorably altered. Therefore, Fere The nci selection mechanism is less effective than the method published herein.

Researchers have used epitopes of unmutated foreign antigens in bacteria. on the surface of a natural bacterium or phage. demonstrated that the epitope was recognized by the antibody. this As such, Charbit et al. (CHAR86a, b) demonstrated that poliovirus vpl The 03 epitope of the protein coated with E, the LamB outer coat tongue of Co11. The C3 epitope is genetically inserted into the bacterial cell surface and exposed to the bacterial cell surface. This was determined from an immunological perspective. Charbit et al. (C) IAR87) similarly , a chimera of LamB and the A (or B) epitope of the p'eS2 region of hepatitis B virus. produced a taupe.

The chimeric LacZ10mpB protein is expressed in E., Col. Depending on the solution, the outer coating or gliding is randomly located (SJLH7 7). The chimeric LacZ10mpA surface protein also contains E, CoII cells. It is expressed and displayed on the surface of the cells (WEINS3). Others are E, coli Type 1 fimbriae (1 (EDE89)) and bacteroid nodular type 1 fimbriae (JENN89). and other bacterial surface proteins expressed and displayed on the surface of the cell chimera. Ru. In the above example, no mutagenized genetic traits have been inserted.

Dulbecco DULB86) transfers heterologous antigen epitopes to viral surface proteins. It suggests processing methods that can be incorporated into quality. Chimeric protein expressed thereby quality is displayed on the surface of the virus in such a way that different epitopes are accessible to antibodies. be done. In 1985, S11ith (SMIT85) Adding a non-functional segment of the lyase gene to gene III of bacteriophage Fl It reports that it was inserted "in phase." Gene III protein is a protein necessary for infection. It is a protein in the inner coat. Smith is a recombinant phage that is EcnR It is adsorbed by immobile antibodies produced against I endonuclease, and also by acid It is demonstrated that it can be extracted by

De’la Cruz et al. (DELA88) are using Plasmodium Fal Circump sporozo-ite protein cycle from Ciparum Seven fragments of the repeat region are inserted into the gene 11 protein of M13. It is said that it is expressed on the surface. They showed that recombinant phage had anti-inflammatory properties in rabbits. These recombinant phages are both antigens and immunogens. It was revealed that it can be used for drawing.

Researchers have shown that similar recombinant phages can be used for T epitope genetic mapping and for vaccines. This suggests that it can be used for development.

These researchers did not suggest mutagenesis of the inserted material, nor did they suggest that the inserted material The injected substance has the ability to specifically bind to receptors other than the antibody's antigen-binding site. Nor is it the perfect binding domain that confers rattan A quality.

McCafferty et al. (MCCA90) reported that the Fv fragment of the antibody 1 protein is expressed by melting to the N-terminus. Fv fragments are mutated Not yet.

Par+1ley and SmithSmlth (PAR is an epitope library can build up all the hexapeptides that can be displayed and the epitope that binds to the antibody. This suggests that they were separated. In the discussion of epitope libraries, the authors There is no suggestion that it is desirable to have a representative list of heterologous amino acids. he Also, the insertion should encode the complete domain of the exogenous protein.

Not discussed. Epitopes are unstructured peptides relative to structured proteins. It is believed that

5cott and 5sith (SCO790) and Cwirla et al. ) created an epitope library. Among them, for the target antibody Potential hexapeptide epitopes encode epitopes and Along with gene III of the phage, the fused gene is expressed in phage-contaminated cells. However, it is assumed that mutations occur randomly by melting of degenerate oligonucleotides. Ru. These cells produced molten phages that displayed epitopes on their surface. solid Phage bound to the determined antibodies were co-eluted with acid. These two studies In order to distinguish the variable region from the wild-type p111 sequence, the fused gene The spacer region formed a coding segment. It changes The changed amino acids are not constrained by neighboring pil+ sequences. Devlin et al. DEVL90) was similarly recognized by streptavidin using M13 phage. A known random 15 residue epitope was selected.

Again, the spacer is a random peptide from the remainder of the chimeric phage protein. was used to remove. Therefore, these documents have mutated It is not discussed that the conformational repertoire of the residue groups (residues, the same shall apply hereinafter) is constrained. stomach.

Another one held by 5cott and Sm1th, Cwirla et al., and Delin et al. The problem is that the collection of amino acid data at each position is very uneven in the library. This is the result of the production of the of degenerate oligonucleotides encoding variable regions. Their biggest concern in the design was that all 20 amino acids could be encoded at each position. The goal was to ensure that Their second concern is maximizing the number of stop lights. It was to be kept to a minimum. Therefore, 5cott and Sm1th as well as CwirIa et al. is NNK (N = equivalent combination of G, A, T, C, K = equivalent combination of C and G) and Devlin et al. used NNS (S=equivalent combination of G and C). most Do not try to reduce the ratio of preferred to least preferred amino acids. Therefore, we did not try to equalize the occurrence rates of acidic and basic amino acids. won.

Devlin et al. developed streptavidin-binding peptides with several affinity selections. peptides, but did not measure affinity coefficients for these peptides. Cwir determined the affinity coefficients of peptides, but the highest hexapeptides (350-300 nM) was disappointing. its parent compatibility, or its “approximate value”, is the natural metto recognized by the target antibody. It was weaker than that of the enkephalin epitope (7nli). Cwir showed that phage-borne peptides with high affinity also bound to acid-eluted folds. I assume that it still exists.

The reason is that the phage (carrying about 4 copies of plII) and the bivalent target This may be due to multivalent interactions between IgG and IgG. Scott and Sm1th have a higher affinity (A2) for the target antibody than the standard myohemelin. A peptide could be found that is equivalent to that of the trine epitope (50 nM). deer However, 5cott and Sm1th probably Some high-affinity peptides were lost due to irreversible binding has expressed concern about this.

La layer et al. (LAM91) is a non-biologically synthesized pentamer on a solid support. Created a peptide library. They have approximately equimolar proportions of lantompe While teaching students to gain access to the world of protein peptides, they also learned about disulfide crosslink formation. Sistine was intentionally left out in order to disappear.

Ladner, GI ick and Bird f08810633 (public Announced September 7, 1988, and also has priority to US Patent Application No. 071021.046. Genex C0rp) (LGB) was used to copy the binding region of the single antibody. The 5CAD/gpV chimera is displayed on the outer surface of the phage. We subcloned the 5CAD gene into the gpV gene of lambda phage and by selecting phages that bind to the antigen using affinity chromatography. , scripts are used to bind various single-span antibody domains (SCADs) to specific antigens. It states that it is OK to do so. The only antigen mentioned therein is the bovine growth host. It's Lumon. other binding substances, targets, carrier organics, and other external proteins. We are not discussing quality.

There is also no mention of the degree or method of mutation.

Furthermore, the exact structure of the melt is not discussed, and the melt that produces good results is not discussed. Also mentions how to identify it and what to do if 5CAD is not revealed. I haven't.

Ladner and Bird f08810661 (published September 7, 1988); - Heavy chain “pseudo-dimerizing” repressor (DNA-binding protein) mutation of the car peptide and subsequent validation for the design of asymmetric repressors. It may be possible to use mutation and selection to create a dictionary of cognitive elements. This suggests that it may be possible to generate it through in vivo selection. The repressor is Not visible on the external surface of the organism.

Substitutes cysteine to promote the formation of disulfide bonds that stabilize proteins. A method for identifying residual groups in the protein obtained is described by Pantogliano and Ladn. er U, S, Patent No. 4.903,773 (PANT90), Panto Meano and Ladner (PANT87), Pabo and 5uchenek (PA BO8B), MAT89, and 5AUE86.

Ladner et al., W090102809, describes how bacteria, phages, or spores Semi of known proteins are revealed as semi-artificial outer surface protein regions of Random mutagenesis (“change”), plus mutants with ideal binding properties describes the affinity selection of Especially for extremely small proteins 10901028 Those mentioned in 09 are Crumbin (3:40.4, 32.16, 26th Sulfides; 46AAs) third region of ovomucoid (8:38.16:35 and 2 4: 56 disulfide; 56AAs) and BPTI (5:55.1438 ．． 30: 51 dislua 4: 513AAs). W0901028 09 also obtains a mixture of all 20 amino acids at that position in approximately equal proportions. It describes methods for "changing" cordons that can be made.

Ba5s et al. (BASS90) introduced human growth hormone into M13 phage gene I. II protein was fused. He said hGH and other "large proteins" suddenly They suggest that it may be possible to mutate and utilize ``bond selection.''

[Summary of the invention] A polypeptide is a collection of like or different amino acids joined by peptide bonds. It is a polymer consisting of main chains. Linear peptide is the main strand of each alpha carphone A large number of different conformations can be picked up through internal rotation around chain bonds. can. These rotations are minimally interfering with glycine and maximally interfering with valine, Isoleucine, especially the proline-bearing side group, is hindered to different extents. It will be done. A polypeptide with 20 residues has 1 determined by various internal rotations. 000 may have a different conformation.

Proteins are polypeptides and are not necessarily located adjacent to a chain It is folded into a well-defined conformation as a result of stability interactions between amino acids.

This sliding structure is usually essential for its biological activity.

For polypeptides with 40 to 60 residues or longer, hydrogen bonds are Non-covalent forces such as coupling, salt bridges, and hydrophobic interactions may cause special bending or conformation. sufficient for stabilization. Are the components of the polypeptide hot or cold? Remains at least in that conformation unless perturbed by denaturing agents such as high pH has been done. The polypeptide then uncurls and "melts" again. peptide The smaller the protein, the more likely its conformation is determined by the environment. Ru. If a small, unconstrained peptide has biological activity, then The tide ligand is a random coil in nature until it comes into close proximity to its receptor. exists in The receptor has unfavorable alternative conformations such as van der faa-1s and others. pep in only one or a few conformations because they are alienated by non-covalent interactions. Accept Chido.

Small polypeptides are less useful as therapeutic or diagnostic agents than large peptides. If the be. In other words, a) good penetration into the organization; b) Fast clearance from the circulatory system (important for contrast agents) C) Low antigenicity Yes, and d) High number of activities Additionally, polypeptides, especially those with fewer than 40 residues, It has the advantage of being obtainable through chemical synthesis. Especially polypeptides less than 30 have priority. In this way, small polypeptides that bind the target of your choice are It would be desirable to be able to utilize a combination of mutation and affinity selection to identify That's true.

However, the majority of polypeptides of this size have no advantage as binding molecules. do not have. According to 01ivera et al. (OLIV90a), [this size Peptides are usually sorted in equilibrium among a large number of conformations (i.e., those with a fixed conformation). (for most proteins, proteins usually have to be larger). ”Target molecule The special bond of the peptide with I need. One residue has three isoenergetic conformations (i.e., heta strands). for decapeptides with de, alpha helices, and Immelmann inversions). There are approximately 6·104 total possible configurations. These conformations constrain Assuming that there is an equal probability for decapeptides that are not affected, only one If a conformation with a probability of The affinity of Tid, if it is constrained to a single effective conformation, is approximately 6. We can expect it to go up by 104. In this way, the design is constrained to a precise conformation. Unconstrained decapeptides exhibit low affinity for cappeptides. It is expected. One of the other conformations of the unconstrained decapeptide is the intended It may be one of the substances that are tightly bound to substances other than the targeted target. This may manifest low specificity. It is a binding site for proteases Through the color system, it is possible to provide proteases with Thus resistance to degeneration may be reduced.

The present invention overcomes the various problems mentioned above and provides a new mini-miniature with desirable bonding properties. Retaining the advantages of small polypeptides by identifying proteins There is. Miniproteins are small polypeptides that are the result of only noncovalent forces. It is too small to have a stable conformation, but it forms covalent cross-links in a stable conformation. (i.e., by disulfide bonds), thereby making it unconstrained. more typical biology of large protein molecules than polypeptides of comparable size. This means that they will have a variety of activities. There are two mini-proteins of particular concern with this invention. It can be divided into types. That is, a) disulfide-bonded microta with 40 or fewer amino acids; protein, and b) a mini-protein with less than 60 amino acids arranged in metal ions. Ru.

The invention is based on genetic mutations that identify novel proteins with desired binding properties. Concerning the structure, expression, and selection of offspring, as well as the miniproteins themselves. and also reveal mini-proteins for potential “target” substances. Regarding the “library” of mutant “gene packages” used to It is something to do. The “target” is often a protein; It doesn't have to be. Targets include not only organic and inorganic substances but also other biological substances. Alternatively, it may be a synthetic polymer.

The previously mentioned patent application W090102809 discloses that stable protein binding is preferred. It may have been mutated to identify novel proteins with specific properties. Generally stated. Appropriate items specifically identified as useful for this purpose. The "parent" protein includes three proteins. That is, BPTI ( 58 residues) (residues, same hereinafter), third domain of ovomucoid (56 residues) , and crumbbin (46 residues). These proteins are 40 to 60 residual groups, where noncovalent interactions between amino acids become significant. have.

These three proteins also contain three disulfons that serve to increase the stability of the molecules. has a field bond.

Nowhere in patent application No. 11090102809 is a polyester with 40 or less residual groups. Regarding peptides, and especially polypeptides with one or two disulfide bonds, The peptide acts as a “scaffold” for mutational fluctuations, ensuring sufficient stability and retention. No specific approval is found for this. These “mini-proteins” , at least, as I have pointed out before, it has great utility value.

The patent application W090102809 also describes different crosslinking formations (Cu:CYS, H This suggests the use of azurin, a protein with I SSHlsSMET). . However, since azurin has 128 amino acids, it is a miniprotein. I can't think of it as quality.

This invention is characterized by the formation of crosslinks regulated by metal ions and the formation of amino acids 6. This relates to the use of mini-proteins of 0 or less.

With this invention, it is possible to specifically bind to targets other than the antigen binding site of antibodies. Protein provided. Proteins can be bound by antibodies, so a simple It cannot be considered a "binding protein". (Please refer to the definition of “binding protein” below) please refer to). The majority of amino acid sequences have more than 6 to 8 amino acids. However, when combined with a single immunogenic carrier, it elicits an immune response and any random Mupolypeptides also have a “binding tag” of minimal affinity and specificity for their substrates. can satisfy the strict definition of "protein". It consists of a large number of random polypeptides only by testing one after the other (and, in the normal case, control the extent and nature, i.e., the linkage with domains with stable structure. Restricting it to residual groups for tensile bonding, the selected residual groups are stable (rather than affecting the binding) this problem is solved. It will be done. Continuing from the above matters (the matters described in "Claims" are not incorporated into this specification) This is a preferred embodiment.

[Brief explanation of the drawing] Figure 1 shows scorpion toxicity (Brookhaven Protein Data Bank registration 15N3) FIG. 2 is a diagram showing the main chain of residual groups 20 to 42. CYS2S and CYS41 are Jisoo Indicating ruphide formation. In natural proteins, these groups are form a disulfide in the chain, but the main chain motion is affected by gamma sulfur. You are obliged to bring the geometry into place. The remaining groups other than GLY are one Heta carbon is marked with a letter coat.

[Detailed description of preferred embodiments] 1. Preface The basic principle of this invention is one of forced evolution. In nature, evolution is The results of genetic variation, beneficial traits, and selected individual reproductive combinations. This trait is what makes the population rich. this invention are mutants of microproteins with dissimilar but related potencies. A complex that creates a single mixture of DNA molecules that coat a specific binding domain. Random mutagenesis of trawled DNA (“change J”) (Variega Genetic variation can be achieved through tion). This invention is desirable Isolating mutated genes reveals novel proteins with binding properties Therefore, the following method is used. That is, 1) the product of each mutated gene is , a replicating gene package (GP) containing a gene (cell, spore, or virus) 2) use affinity selection, i.e. Then, using the selection to bind to the target material, for packages containing genes that specify proteins with improved binding properties. This increases the population of packages. Finally, the increase is A package containing a gene bound to a target for reproductive purposes by a protein This is achieved by allowing only Evolution is driven by selection for target substances. It is "compulsory" because it is provided to the public, and the special cordon is provided to the It is ``forced'' because it mutates at a higher rate than the number of times it occurs.

The revealing strategy is, firstly, that an affinity molecule (which may be an antibody) is available. . A stable, structured domain (“initial potential binding domain”, IP BD) by changing the genetic package to reveal it. change success depends on whether the altered genetic package binds to the affinity molecule. It is easily measured by determining the

The IPBD was chosen because it allows extensive mutagenesis. IPB D is displayed on the surface of the package, subjected to affinity selection, and encodes a gene. It has been shown that PBDs are subject to a specific pattern of multiple mutagenesis. Are known.

Variation, as used here, refers to each single point. Genetic package revealing a temporal binding domain (IPBD mutant) This leads to the group production of Ji. However, although the "changes" are heterogeneous, Overall display of a group of potentially related binding domains (PBDs) do. Each genetic package has a PBD exposed on the surface of its specialized package. A 1° affinity selection carrying a version of the pbd gene that coats the desired to identify genetic packages harboring PBDs with binding properties of These genetic packages can also be amplified. For affinity selection and amplification After one or several cycles of isotopic enrichment, the successful binding domain (SBD) , ) is recovered from the selected package.

If necessary, DNA from the SBD support package [parental potential binding Using the SBD of the final round of changes as domain J (PPBD), PB "Change" to the next generation of D. Then, repeat the operation until you get a satisfactory result. The work can continue. IPBD<! :Structural and evolutionary differences between the first generation of PBD. Because of the relationship, rPBD also has a “parental potential binding domain J (PPBD)”. It is considered.

When a microprotein is altered, the residual groups covalently crosslinked to the parent molecule are unchanged, thereby stabilizing the conformation. For example, microproteins and Since some cysteines remain unchanged in the form of combined disulfide changes, , under the conditions of expression and display, in a covalently cross-linked form (i.e., if one cysteine (disulfide bonds) between several pairs, and a highly fluctuating linear The conformation adopted by the intermediate amino acids is well constrained. Don't paraphrase If the constrained scaffold is incorporated into extensively randomized polypeptides, .

Once a microprotein with the desired binding properties has been characterized, it is It can be reproduced not only by DNA technology but also by non-biological synthesis methods.

For the purposes of the above claims, protein P is defined as "binding protein". ” and at least one molecule and ion other than the variable domain of the antibody Or, for atomic species A, the dissociation constant Ko (P, A) < 10-' mole s/1 iter (preferably <10-'+oles/1 iter).

“Variable domains of antibodies” in (1) above is intended for clarity. , one protein is a "binding protein" simply because it is an antigen. ” is unthinkable.

The largest type of protein is a distinguishable spore called domain (RO3S81). is incorporated within. Protein domains have been defined in a variety of ways. . The definition of "domain" that is important for stability is -1 high temperature or chaos. Lopik (chaotr.

pic) An m-preference that holds the entire structure on the surface of a disturbing force like an agent. Although intentional, the homology of atomic coordinates and protein sequences cannot be completely ignored. stomach.

A protein whose domain specifically binds to a selected target. When taking primary responsibility for quality capabilities, it is here It will be called (BD).

“variegated DNA (vgDNA) ) is a mixture of DNA molecules of equal or similar length; If this occurs, each codon encodes multiple different amino acids. Although it varies in several codons, it is possible to place a single amino acid in the position of other codons. Coat. In engineered DNA, the codon is variable and The regions and frequencies of different amino acid occurrences coated by variable codons are is known to be predetermined by the synthesizer. Ta Even if the individual DNA molecule sequences in that mixture are one a priori (prior In order to know i), one must not allow others to know how to synthesize it. The number of designated variable codons of the designated DNA is preferably 20 or less, The most desirable number is 5 to 10. Amino acids coated in each variable codon The mixture differs depending on the cordon.

A population of genetic packages into which modified DNA has been introduced can be said to have been "modified." Ru.

For the purposes of this invention, "potential binding protein" (PBP) refers to proteins coated by one species of DNA molecule in a population of DNA refers to quality. The area of variation within it is the binding domain for the target material. one or more polypeptide segments that have the potential to act as a main occurs when one or more subsequent elements are coded.

[Chimeric protein J is the first protein The amino acid sequence has a domain different from that of the first protein, and the first A second amino acid that defines a domain that is not sufficiently homologous to the protein It is to melt with acid sequence.

A chimeric protein is found in one organism displaying the first protein. (also found in heterologous proteins, representing a heterologous domain) Maybe. Or it is a protein structure exposed by a foreign organism. Zozo's ``in-terspecies'', ``ash amount J'' neric) and other melting.

The amino acid sequence of one of the chimeric proteins involved in this invention is defined herein. “Typically derived from the outer surface protein of gene package J (GP). One that shows PBD on the surface of is GP (PBD). manifested in a single In this case, the second amino acid sequence has the characteristics of a protein (or domain). However, although it is incorporated into the chimeric protein as a distinguishable domain, There is one. It consists of a first amino acid sequence (with an intervening spacer) or without) at the amino or carboxy terminal of , or interrupt the first amino acid sequence. The first amino acid sequence is a gene package corresponds to or is regulated by a surface protein of the surface of the page. i.e. join This is to make it easier to reveal the domain.

Il, micro and other mini-proteins In this invention, disulfide-bonded microproteins and metal-containing miniproteins are as an initial potential binding domain (IPBD) to verify the revealing strategy. , and actually hope to obtain a BD with the desired target binding characteristics. PPBD is used as a binder. Unless otherwise specified or depending on the context Unless required, references to IPBD are mutatis cutand is, and the same applies to PPBD.

For the purposes of the claims that follow, the microprotein is approximately 6 It has 40 residual groups from the base. Microproteins are sub-groups of mini-proteins. sets, which have no more than approximately 60 residual groups. microtan Since proteins constitute a subset of mini-proteins, they are conveniently referred to as mini-proteins. The term fowl refers to disulfide-bonded microproteins and metal regulatory miniproteins. Both will be cited as necessary. I PBD had known binding activity is a mini-protein or has no known binding activity, but have a second or higher contributing structure (tears, grooves, etc.) It is one of the things that I If the PBD has known binding activity, the target It is not necessary to have a special affinity for the target substance. IPBD is a naturally occurring It is not necessary that the miniprotein and its sequence are identical. It is a known mi It may be a ``homologue'' with an amino acid sequence that is ``sufficiently equivalent'' to the sequence of the 2-protein protein. I don't know, or maybe it's completely artificial.

To determine whether sequences are considered "sufficiently equivalent," It is necessary to consider the following matters. That is, if the array best fits according to the standard arithmetic rules. degree of sequence similarity, cross-linking (i.e., disulfide similarities in the connectivity patterns of fido bonds), X-ray diffraction analysis or NMR Therefore, when displayed, the degree to which proteins have similar three-dimensional configurations, and their Among serine protease inhibitors, proteins that are arranged as , there is a set of members whose sequences are 30% homologous and are recognized as homologs. There is a group of proteins that have been

Candidates for IPBD must satisfy the following criteria. That is, 1) The domain remains stable and exists under the conditions of intended use (domain includes all of the inserted proteins.

That is, alphaconotoxin G I (OLIV90a) or CM TI-11I (MCfH89), 2) Knowledge of amino acid sequences is difficult to obtain. and 3) have a special and high affinity for IPBD. molecules are available. This is abbreviated as AfM (IPBD) .

A single species of molecule that has affinity for IPBD (AfM(IPBD)) If the genus were available, it would be used for the following purposes. That is, a) Detection of I PBD on GP surface, b) M) Display level of affinity molecules on IJ Fuchs and C) determining the efficiency and sensitivity of the affinity separation procedure. AfM If two types of (IPBD) are available, one is expensive and the other is expensive. selects an affinity for an intermediate IPBD. Species and genera with high affinity are It will be used for initial detection and to determine efficiency and sensitivity. and the middle Species and genera with affinities will be used to inform optimization.

I If the PBD itself is not a known binding protein, or If the natural target is not purified, antibodies made against IPBD may It may be used as a compatible molecule. The use of antibodies for this purpose is should not be considered the final target.

There are many (IPBD candidates) for whom all the above information can be obtained. However, it is reasonably practical to obtain one. For example, CMTI-111 (29 residual Group>(CMTI-type inhibitors include 0TLE87, FAVE89j lEC8 5, W I EC85, MCWH89, BODE89, HOLA89a and b heat-stable enterotoxins (E, col) i5T-la) (18 residues XGUAIl189, BHAT86.5EX18 5, S old M87, TAKA85. TAKE90.780M85a and blYOS) 185, DALL9 [1, DWAR89, GARI87, GUZM89.611 2M90, HOUG84JtlB089, UPE9G, OKAM87, OKA M88, and OKAM90), alphaconotoxin Gl (13 residues) (H AS1185, AIJQ89), muconotoxin GIII (22 residues) ( HID090), and Konas King Kong microprotein (27 residues) (voOD90) is included. Information regarding the composition can be obtained from X-ray or Nutro diffraction studies, NMR1 chemical drug cross-linking or Rahel, related proteins It is a model from a known structure or a logical calculation. 3D composition information is obtained by X-ray diffraction , neutron diffraction or NMR can be recommended. The reason is that these methods are Almost all of the atoms can be located within the defined limits. Table 50 is desired. Some of the IPBDs that can be used are listed below.

Mutations may reduce women with PBD. Therefore, the selected IP BD should preferably have a high melting temperature, i.e. at least 50 degrees Celsius. It is desirable that it be stable over a wide pH range. That is, 8. A value between 0 and 3°0 is preferred, and a value between 11.0 and 2.0 is most preferred. in addition Therefore, 5BDs are extracted from the I PBD selected by mutation and are bound to each other. This selection retains sufficient stability.

Preferably, an alternative to the IPBD that produces various PBDSs is The melting point of the substance should not be lowered below approximately 40 degrees Celsius. One mini protein It is useful to have a covalent bridge such as a disulfide or more. standing and they will ensure sufficient stability.

In vitro, disulfide bridges are naturally formed in polypeptides as a result of air oxidation. can be formed. What happens inside a living body is, of course, more complicated than that. very few cells The protein within has disulfide bridges, perhaps because it is in a strongly reducing environment This is probably because it is retained by the glutathione system. dithrid bridge are normally present in proteins that move around, or in snake toxins and other toxins (e.g., conotoxins, charybdotoxins) in) bacterial enterotoxins), peptide hormones, digestive enzymes, and supplemental nutrients. proteins, immunoglobulins, lysozyme, protease inhibitors (BPTI and Homolobes, CMTI-+11 bita w+axima]) Lipsin inhibitor III) and its homolobes , hirudin, etc.) and proteins in milk that move within cells. It is present in proteins that

The disulfide bonds that close the loops of tough cellular white rust are linked to pepsin, thioredoxy Insulin A chain, silk fibroin, and lipoamide dehydrogenase has been discovered within. The bridging cysteine residues are present in the polypeptide chain. separated by one to four residual groups along the model building, X-ray diffraction analysis, and NMR studies reveal the alpha carbon path of such a loop. shows that it is usually flat and robust1, There are two types of disuride bridges in immunoglobulins. One is preserved cell white It has 60 to 70 amino acid residues and is responsible for almost all immunoglobulins. is repeatedly found in the following domains. There is a deep gap between competing beta sheets. These buried bridges are protected from solvents and in the presence of modifiers. Can only be normally redeemed. Other disulfide bridges are mainly intracellular bonds. , present on the surface of molecules. These bridges are solvent accessible and relatively easily returned. (STEI85). Microprotein dispersion according to this invention Rufid's bridge is a white rust link between cistines that have a smaller tread interval. It is.

If a microprotein has multiple disulfide bonds, at least Preferably a swarm of two cysteines. i.e. they are directly adjacent to the chain adjacent or separated by a single amino acid <-C-X-C-) is desirable. In either case, the cluster of two cysteines is called steric hindrance. It is impossible to pair with each other for a reason.

Then some realizable phases are reduced.

A polypeptide with 16 residues that links 3 to 8 amino acids If the silver disulfide bridges have a span of 4, they have a span of 7. Form a second span. In both, four cysteines bind the polypeptide to four cysteins. Divided into internal segments (1-2, 5-7, 9-11, and 13-16) Ru. (Observe that there is no segment between Cys3 and Cys4. please. ) The pattern of cross-linked microprotein connections is This is a simple representation of the relative position of the ends of the blocks. For example, two disulfide bonds For a microprotein with , the connection pattern "1-3.2-4" is It means that. That is, the cysteine of the first cross link is the cysteine of the third cross link. a disulfide bonded to the cysteine (in the first sequence) of the slink, The cysteine of the second crosslink is combined with that of the fourth.

The degree to which cross-links constrain the conformational freedom of miniproteins and whether they The degree to which a protein is stabilized may be estimated in a variety of ways.

They include the following methods. i.e. absorption spectroscopy (amino acids buried) (detecting whether proteins are exposed), circular dichroism (detecting helical content of ), nuclear magnetic resonance imaging (provides a general picture of several nuclei in a special chemical environment), (simultaneously showing nuclear mobility), X-ray or nuclear This includes tron diffraction analysis. The stability of mini-proteins depends on temperature, pH1 and other factors. As a function, protein assembly can be determined by monitoring changes in absorption at various wavelengths. You will be exposed when you are freed from entrapment.

Similarly, the release of the miniprotein as a result of the denatured state is consistent with NMR line positioning. Bring about a change in width. Circular dichroism (CD) spectra are extremely sensitive to conformation It is.

The modified disulfide-bonded microprotein according to the present invention is divided into classes. That is, class 1 milliloproteins have disulfide bonds. It is characterized by a pair of cysteines that can interact to form bonds. and the bond has a span of approximately 9 residues or less. This disulfide The bridge preferably has a span of less than two residues. , which is a geometric function of disulfide. Residues with two or three spacings , one residual group is desirably such that the upper limit of bridging residues is It doesn't have to be very precise, but generally speaking, the larger the spacing, the more disulfide bonds there are. There are fewer conformational constraints imposed on the linear intermediate amino acid residue by the combination.

The main chain of such a peptide is allowed extremely little freedom, but no tension. Ji The free energy released when sulfide is formed is accompanied by cysteine. The free energy lost by the main chain when confined in the conformation that brings It exceeds. Due to the loss of free energy during disulfide formation, The proximal ends of the site groups maintain a fixed relationship to each other. target When bound to the domain, the domain has free energy to enter the correct conformation. There is no need to consume. The domain is in the other conformation and the target is It cannot be combined with something that does not exist.

Disulfide bridges with spans of 4 to 5 are particularly preferred.

As the span increases to 6, the effect of the restraint decreases, in this case at least Preferably, one of the residues is an amino acid that imposes constraints on the geometry of the backbone.

Proline may impose the greatest limitation. Valine and isoleucine are less restricted by the backbone. limits may be imposed. The preferred position of the non-cysteine residue imposing this constraint remains unchanged. It is adjacent to cysteine. However, it was like building a bridge. It may be one of the remaining groups. If the span is 7, the main chain conformation is restricted. It is desirable to add two amino acids that are These amino acids are in the 7th position Any location is fine. But preferably two bridges directly adjacent to the Sistine It is a residual group bridging the . If the span is 8 or 9, the constraint The number of amino acids may be increased.

Although class I microproteins can hold up to 40 amino acids, The number is preferably 20 or less.

Class of Disulfide Bonds ■ Microproteins are exposed to solvents. for that, We must avoid exposing the altered population of GPs. Replacing the GPs The collected population reveals class I microproteins in a disulfide-destroying reagent solution. Show.

Class rlE cloproteins contain a single distinct protein with a span of nine or more amino acids. It is characterized by a ruphide bond.

The bridging amino acids form secondary structures that help stabilize the conformation. Ru. Preferably, these intermediate amino acids are formation of a supersecondary structure.

-Cys-alpha hel 1x-turn-beta 5trand-Cys-r −−−−−−−S −−−S −−−−−−−−−−−−−1−Cya− alpha helix-turJ'I-alpha helix-Cys'---- -----s----s-----------.

-Cys-beta5trand-turn-beta5trand-Cys-known Based on research on proteins, alpha helix, heta strand, or a special residual group found in the Immelmann reaction, or a special cippetite. Or the tripeptide propensity can be calculated. Among these secondary structures Typical occurrences of amino acid residues are listed in Table 6-4 of CREI84.

For a detailed treatment of predicting secondary structure from amino acid sequences, see 5CH See Chapter 6 of U79.

In order to design an appropriate hairbin structure, it is necessary to use a protein whose three-dimensional conformation is known. The actual structure can be copied from the texture, and the data on the number of occurrences can be used to You can also design by combining the above two methods. Ru. Preferably, one or more actual structures are used as models; The data on the number of occurrences can be used to determine the structure that can be manufactured without disturbing the structure. It is used for.

Preferably, no more than three amino acids are present in cysteine and alpha helix or helix. - Intervening between the beginning or end of a tastrant.

More complex structures (such as double hairpins) are also possible.

Class IIra microproteins are characterized by two disulfide bonds. Ru. These are also optionally described in relation to the class IT microproteins mentioned above. It is characterized by a secondary structure. Three phases are possible for two disulfide bonds. It is Noh. Multiple disulfide bonding possible phases are thermally stable if desired Reduced by a group of cysteines present in the enterotoxin 5T-TA It might happen.

Class l1lb microproteins contain three or more disulfide bonds It is characterized by And preferably, as mentioned above, at least when It is to have a sting.

Metal finger mini protein. This invention also provides chromatography through disulfide bonds. It is related to mini-proteins other than Slink. i.e. finger tongue It is an analog robe with a high quality.

Finger proteins are characterized by their finger structure. Its structure In the structure, two Cys and two Hi metal ions form a tetrahedral arrangement. It is controlled by the s residue group. The metal ion is zinc (II) Primarily, but sometimes iron, copper, cobalt, and others. "fin "Gar" is a large number of sequences (Phe or Tyr) -(IAA)-Cys- (2-4AAs)-Cys-(3AAs)-Phe-(5AAs)-Leu-( 2AAS)-His(3AAS)-His-(5AAS). (BER G88. GIBS88). Finger proteins are typically finger mochi. It has many repeats of fingers, and one finger is activated in the presence of zinc ions. It is known that it is woven into (FRAN87, PARR88). two Whether two fingers are necessary for DNA binding is still a matter of debate. This invention , which involve mini-proteins with one or two fingers.

Other combinations of side groups include cross-links related to polyvalent metal ions. It leads to formation. For example, Sumilers (311MM91) One 18-amino acid miniprotein is the capsid of HIV-1-Fl. id) reported that it was discovered in proteins. it is bonded to the zinc atom It had three cysteines and one histidine. Target is not necessarily a nucleic acid You should understand that it doesn't have to be.

PBS adjusted to Some enzymes that can selectively modulate certain sidegroups of the proteins listed below. and chemical reagent solutions. They are a) protein-tyrosine kinase, El1 mans reagent, methyltransferase (side group of methylate GLU) ), serine kinase, proline hydroxyase, converts GLU to GLA Contains vitamin dependent enzymes, maleic anhydride, and alkylating agents. this of a modified population of GPs (PBD) with one of the enzymes or reagent solutions from Handling adjusts the side groups affected by the chosen enzyme or reagent solution do.

Enzymes or reagent solutions that do not destroy GP are highly desirable. Saidog Such adjustment of the loop directly affects the binding properties of the revealed PBDS.

Utilizes affinity separation methods to bind to predetermined targets. We are trying to strengthen our GPs. All active binding domains are genetically defined. Since it is not fixed, it is necessary to repeat the adjustment after morphogenesis at each reinforcement stage. be. This method envisions the chemical synthesis of these 5BDSs, so mini- It is particularly suitable for protein IPBDs.

III. Change Tactics - Obtaining Potential Binding Domains with Desired Diversity Mutagenesis 111, A. General Largely different amino acid sequences can be obtained by mutation of several domains; Forced when compared to several different domains that expose and humiliate in discoverable quantities. The effectiveness of evolution can be largely determined by careful selection of residue groups. (to be raised. First of all, Known tags that can affect binding activity (i.e., residual groups on the surface) Residue groups of proteins and known proteins that do not unnecessarily reduce their stability. The residual groups were identified. All or all of the cordons coating these residual groups Some of them are changed one after another to create populations with changed DNA. One by one A group of residual groups that is close enough to the surface to touch one target molecule is , is a set prepared for simultaneous changes. There are various groups of people whose DNA has been changed. It is used to reveal a potential binding domain. and the tag you are interested in The ability to connect with the target is evaluated.

The method according to the invention, as described above, furthermore produces and highly convertible in terms of the nature of the population and the selection of novel binding proteins. , is outstanding compared to other methods. We have the revealed potential binding domain adjacent amino acid sequences that are related in an effective and organized manner Forces the ``array space'' to be extracted. Four goals are used here: leading to various changes. Preferably, 1) the majority (i.e., 107) of the variants are available; 2) a change in the likelihood of a high rate of return occurs in a detectable amount; 3) it is desirable. 4) the frequency of occurrence of the change is relatively constant, and 4) the variation is limited to a limited number of Occurs only in amino acid residue groups. Most preferably, the fluctuations are in the potential coupling domain. occurs in residual groups with side groups directed toward a common region on the surface of the It is.

This method uses indiscriminate chemicals such as radiation and hydroxylamine that modulate genes. It should be distinguished from the simple use of natural mutagens. indiscriminate sudden change inducer In use, the site of mutation is uncontrolled and highly biased. Ru. Many mutations affect residual groups that are not part of the binding domain. Ru. If a chemical mutagen is directed throughout the genome, most mutations , occurring in other genes than the one encoding the potential binding domain. Ru. Moreover, at a reasonable level of mutagenesis, the adjusted codon is limited and unbalanced as it can be characterized by one base change. Only the realm of possibilities that can be explored is explored. Similarly, remote is random Special site targeting using oligonucleotides as mutagens with unique sequences This is the use of natural mutagenesis technology. These specialized technologies are used in the production and testing of numerous variants. Not involved. The expertise in random mutagenesis that is attracting attention is unknown. However, the importance of controlling the fluctuation distribution is overlooked.

The potential binding domain is first designed with amino acids. once, which Once we identify which residue group is mutagenized, we can determine which mutation is at that site. Once the process is identified, the altered DNA encoding the various PBDs can be identified. can be designed. coated PBD, the PBD becomes the target. having an affinity for a target guarantees a reasonable probability that it will be detected. Ru. Of course, the large number of independent transformants and affinity separation expertise available It would impose limits on the degree of change that is possible within a single round of change.

There are various ways to create diversity in proteins.

(Refer to R1 (H86, CARU85, and 0LIP86)) One The extreme is changing just a few residue groups in as many proteins as possible. That is. (among others, CARU85, CARU87JICH86, and f) See lAR86. ) This method is called “focused mutagenesis” (Focus ed Mutagenesis). Typical “focused mutagenesis” The strategy is to -take out the residue groups from sets 5 to 7 and select the possible residue groups from 13 to 20. Change each person with their potential. Another plan for mutagenesis (“diffusion mutagenesis”) is A more restricted set of choices results in a larger number of residues being changed.

(See VER386a and PAKU86.) Adopted changes (varie The ``gation'' pattern lies between these extremes. That is, 2 Two residue groups are exchanged through 0 amino acids, leaving only two possibilities. Two more amino acids were changed, and in the fifth, 10 of the 20 amino acids were changed.

There is no limit to the number of cordons that can be exchanged at the same time. However, a possible PBD arrangement If the sequence is truly manifested by at least one genetic package, then It is desirable to employ mutagenesis strategies that produce reasonable probability. Delightfully are muteins and muteins encoded by VgDNA. The mutin consisting of the least preferred amino acid at the selected site is The probability of being represented by at least independent transformants in the library is small. It is at least 0.50, preferably 0.90. (more preferred amino Mutins consisting of acids can, of course, occur in that same library. be. ) Preferably, the changes involve no more than 10 different DNA sequences (more preferably, Typical amino acid sequences exhibiting different 106-107 amino acid sequences The aim is to provide a population of transformants with a large population.

Alpha helis (hel1ces) and beta strands Class ■Microproteins that do not have any mutations preferably each replaces 4 to 8 non-cystine cordons, thereby Thus, each of them encodes at least 8 of the 20 positive amino acids. That's true.

Each cordon change can be changed specifically for that site. Preferably, cysteine is Not one of the potential substitutes, even if cysteine was among them Even if.

Typical transformation tactics when the miniprotein is a metal finger protein In , two Cys and two HiS residues and an optional front The Phe/Tyr, Phe and Leu residues mentioned above are unchanged, and A plurality (usually 5 to 10) of the other residual groups may be replaced.

Microproteins contain one or more alpha-helis and beta-strands If the type is characterized by A set of potential amino acid adjustments disrupts secondary structure at that site. It is desirable to incorporate things that do not. Number of possibilities for each variable amino acid Since the number of variable amino acids is limited, the total number of variable amino acids changes the effectiveness of selection in the selection process. It will grow without any problems.

For class III microproteins, preferably 20 or less, more preferably In other words, 5 to 10 cordons change. However, diffusion triggers If quality is used, the number of changed cordons can be large.

Changes usually involve substitution of one amino acid for another in the specified variable codon In such cases, it may be related to insertion or deletion of amino acids.

111, B. Identification of residues to be replaced Let us now consider the principles that help select the residual group of the I PBD to be replaced. foundation The theory is that only structured proteins exhibit special binding. Ru. In other words, the above-mentioned protein is a special chemical substance to the exclusion of the majority of others. Can be combined. , in this way, the residue group replaced is the basic I It is selected with the purpose of preserving the PBD structure. The PBD is closed from the sliding structure. Alternative substances cause GPs to carry genes that bind indiscriminately; They can be easily removed from the population. Amino acid substitute exposed to solution has little effect on the three-dimensional structure of the substitute material at the internal locus.

(PAKU86, REID88a, EISE85, SCH■79.169-17 1 page, and CREJ84, 239-245.See pages 314-315. ). Internal residues are often conserved and amino acid types vary from protein to protein. can be meaningfully replaced with a different type without the risk that the structural structure may be destroyed. It is impossible. However, ■ Mustard or F to Y Conservative changes in internal residues are missed. Such conservative changes may affect adjacent proteins. It subtly influences the arrangement and dynamics of the quality residues, and such "delicate regulation" ” becomes useful once the SBD is found. Insertions and deletions are more routine than anywhere else. more easily tolerated within the group. (THOR88).

Data on IPBD and the residue groups that change during the conversion cycle are determined. Data about the target that can be helpful in determining the target include: In other words, 1) structure, also or at least a list of residual groups on the IPBD surface; 2) homologous to the IPBD; 3) a model of the target molecule or a list of target sequences; This is Tandoin.

111, C, Determination of Alternative Set for Each Parent Residue Selecting Residues for Replacement The region of amino acids allowed for each variable residue is then determined. all levels of change is the number of variants in each changed residue. Each deformed residue has 2 It produces 20 different potential substances and has different schemes of transformation.

Amino acid set potentially encoded by a specific altered codon is called the "alternative set".

A computer that controls the NA synthesis agent, such as Millige Roa 500. The computer contains some nt-based (i.e., nt phosphoramidite-phor phoram-idites) to create two or more reservoirs ( The base of the oligo nt, which has the role of nts allocation, is combined from can be achieved. Alternatively, nt bases can be mixed in other proportions and ” into one of the reservoirs for synthesis. Each nucleo of the cordon The “mixture” of substrates at the cord site is the Determine the relative frequency of occurrence of the amino acids.

Easily altered codons have degenerate nucleotide sites and are not homologous. It is obtained from a mixture of two or more groups mixed in a ratio of 1 to 3.

These mixtures are clarified by standardized "ambiguous nucleotide" codes. It is stated in the detailed description. In this coat, for example, degenerate cordon rsNTJ ``s'' is an equimolar mixture of G and B groups, and rNJ is an equimolar mixture of all four groups. is a mixture, and ITJ is a single invariant group, thymidine.

A complexly modified cordon is an equimolar mixture of two or more base materials. At least one of the three sites is filled with one base material that is not It is something.

Desired alternative set for both easily and complexly modified cordons. can be made. The special amino acid or amino acid class is appropriate. If there is no information showing the mini-proteins above the level that can be found. Since labeling is uneconomical, efforts are being made to substitute all amino acids with equal probability. Ru. The equivalent amount of four nts in each site of one codon (NNN) is produces an amino acid distribution proportional to the number of codons coated by amino acids . This distribution gives rise to two basic residue groups for one acid residue. have advantages. Furthermore, RlS, which is six times as large as W or M, causes L. do. If five codons are synthesized with this distribution, some of L, R1 and S Each of the 243 sequences encoding a combination of W and M 7776 times more abundant than each of the 32 sequences encoding the sequence. Les that can be discovered In order to have 5 WS presents with Bell, each fold must be 7776 or more. (L, R, S) must have an array present.

Special amino acid residues define the three-dimensional structure of a polypeptide in several ways. 1. Flexibility of the backbone of the polypeptide b) add hydrophobic groups; C) add charge groups; d) allow atomic bonds; e) Chelation with disulfide, metal ion, or biosurrogate device group form cross-links such as bonds with groups.

Lundeen (LUND86) is a protein known for its three-dimensional structure. Frequency of helial, beta-strand, turn, and coil amino acids present in Create a table and distinguish between CYSs and semi-cystines that have free thiol groups. Separated. Free cYs are most numerous (found in helices), and half-cystine It has been reported that the largest number of these were found in the However, hemicystine It is regularly found in Pease et al. (PEAS90) reported that two cysts constructed a peptide with One end of each has a true stable alpha lip. It's in Kusu. Avamin has a similar structure. (lEMM83.PEA S88).

Flexibility: GLY is the smallest amino acid with two hydrogens attached to C1a. GLY is Since it does not have C<-2, GLY leaves maximum flexibility to the main chain1, thus , C, LY frequently occur in Immelmann inversions. P　R01ASP, It occurs particularly frequently in 5ER1 and THR.

ALAlSER, CYS, ASP, ASN, LEU, MET.

Accessories such as PHE, TYR, TRP, ARG%HIS, GLU, GLN and LYS etc. Mino acids have unbranched beta. Of course, the side group SER,A SP and ASN frequently form hydrogen bonds with the main chain, thereby making them can add undesirable conformations to the main chain. VAL, ILE and THR is a branched beta carbon that makes the extended backbone conformation more desirable. have. In this way, VAL and ILE are the most frequent among the beta sheets. seen in The side groups of THR can easily form hydrogen bonds with the main chain. Therefore, there is less tendency to exist in the beta sheet.

The proline backbone is particularly constrained by cyclic side groups.

The phi angle is always close to 60 degrees. The majority of prolines are found on the surface of proteins be done.

charge LYS and ARG are respectively lo, and 4 is also L<12. Single positive charge in nor H below o Hold the load. In any case, methylene glycol with 4 and 5 amino acids respectively The loops are capable of hydrophobic interactions. ARG's guanidinium group is The amino acid group of LYS can contribute only three hydrogens at the same time, but five of hydrogen can be contributed at the same time. Furthermore, the geometry of these groups are different, so these groups cannot sometimes be interchanged.

ASP and GLU retain a single negative charge at pH above -4, 5 and 4.6, respectively. do. Since ASP has only one methylene group, some hydrophobic Interaction is possible.

The geometry of ASP plays a role in forming hydrogen bonds with backbone nitrogens.

The nitrogen is consistent with ASP, which is frequently found in Immelmann inversions, and At the beginning of the squirrel. GLU is frequently found in alpha helis, especially in this It is found at the terminal amino acid position of L. The reason is that the negative charge of the side group This is because the charge has a stabilizing interaction with the helical dipole. (NICH 88,5ALI88).

HIS has an ionization package in the physiological region, viz. 6.2 There is. This package can be used in close proximity to charged groups or It can be changed at a location close to the person or recipient. HIS contains zinc, copper, and iron. It can bind to metal ions such as

Hydrogen bond/ Besides the charged amino acids, SER, THRlASN, GLN, TYR and T RP can participate in hydrogen bonding.

cross link The most important loss-linking type is the disulflink format between the CYs residue groups of the thiol. It is a fido bond. In a suitable oxidizing environment, these bonds form spontaneously.

These bonds make specific conformations of proteins or miniproteins very stable. It can be made into Oxidized thiol reagent solution and reduced thiol reagent solution When a mixture of drug solutions exists, a transformation that allows the most stable conformation to prevail takes place. be exposed. Teha, KATZ90J regarding proteins and peptides ATS89. PERR84, PERR86,5AIJE86, WELL86, J ANA89. Please refer to HORV89, KISH85, SOLT5CHN86 and.

Pond crosslinks, which are formed without the need for special enzymes, are as follows: . That is, 1) (CYS) 4: F e Rubretoxin (CREI84.376 page) 2) (CYS) 4: Zn aspartate transcarbamylase (CRE 184.376 pages) and Zn-finger (HARD90) 3) (HTS) 2 (MET) (CYS): Cu Azurin (CREI84.376 pages and basic “ Blue JCu cucumber protein (GUSS88) 4) (HIS), :Cu CuZn peroxide dismutase 5) (CYS) 4: ( Fe4S4) 7 Eredo t-shin (CREI84.376 page) 6) (CYS)=　(HI　s)2:　Z　n　Annine-Finger(GIBS) 88.311MM91) 7) (CYS)s(HI　S): Zn anine-finger (GAUS87, GIBS88) (Hls) 2 (MET) (CYS): The cross link with Cu is HIS and MET have the potential advantage of not being able to form cross-links without Cu. have.

Easily replaced cordon The following easily substituted codons indicate that they have a relative balance of amino acids. This is useful because it encodes a set of That is, 1) Coat the following set (L, P, H%R, V, A, D, G) with SNTI; , i.e. a) one acidic substance (D) and one base (R) b) aliphatic (L, V) and aromatic Both the aqueous (H) large (L, R, H) and small (GlA) side groups d) Rigid (P) and flexible (G) amino acids e) Amino acids once encoded 2) RNG codes the following set (MST, R%V, A%E, G), all Namely a) one acidic substance and two bases (not the optimal one but the acceptable one) b) Hydrophilic and hydrophobic substances C) Amino acids once coated 3) RMG coats the following set (T, with %A,E), i.e. a) one acidic, one base, one neutral hydrophilic b) three desired a Rufaheris C) Amino acids once coated 4) VNT is the following set (L, PSH, R, I, T, N, S.

V, A, D, G), i.e. a) one acidic substance and one base vinegar b) All classes: charged substances, neutral hydrophilic substances, hydrophobic substances, rigid substances and flexible substances, etc. C) Amino acids once coated 5) RR3 coats the next cent (N, S, to R, D, E, G2), e.g. Wachi a) Two acids and two bases b) Two neutral hydrophilic substances C) Single grisidi coded twice 6) NNT is the next cent (F, S, Y, C, L, P, H, R1■, T, N, V, A, D, G), i.e. a) provides 15 different amino acids 16 DNA sequences.

Only serine is repeated, the others are present in equal amounts. (This is the library pole Allow effective extraction at any time. ) b] These are the same number of acids and base amino acids. Yes (one addressed to D and R) C) All major classes of amino acids are present. i.e. acidic, base, fatty hydrophobic aromatic hydrophobic substances, aromatic hydrophobic substances, and neutral hydrophobic substances 7) NNG is the next noset (L2, R2, S, WSPSQ, M, T.

K, Vt A, E, G, stop), i.e. a) alpha helis (for L, M, A, Q, E, and to a lesser extent S, R, T) Fair and dominant of conformation-favoring residue groups b) Coat 13 different amino acids. (VHG coated by NNG code a subset of the given set.

It has equal acid and base, 5 out of 9 prefer alpha helix. It encodes nine amino acids found in nine different DNAs. ) For initial changes, NNT is also preferred in the majority of cases. However, amino If the cordon coats the acid incorporation into the alpha helix, N NG is preferred over NNT.

Below we analyze a few simple changes in terms of efficiency. Thereby, the library – can be extracted.

A library of random hexapeptides coated with (NNK)' has been reported. I was warned. (SCOT90.C1111R90). Table 130 shows such Indicates the expected behavior of the library.

N N K Ha, PHESTYR%CYS1TRP, Hls, GLN.

ILE, MET, ASN, LYS, ASP, (-7GLU (full facet) Create a single coden for VAL, ALA, PROlTHR, and GL Create two cordons for each of Y (Fufuy set), LEU, ARG , and 5ER (omega set).

According to the number of amino acids in each of these sets, as shown in Table 13OA. Thus, the 64,000,000 possible arrays were divided into 28 classes. largest class is a phiomega four algorithm with a possible sequence approximately equal to 14.6 percent. It is Fa. Apart from selection - all sequences of classes have the same probability of being produced. have. Table 130B shows arbitrary DNA taken from the (NHK)6 library. probability that a sequence encodes a hexapeptide belonging to one of the defined classes It shows. Only 6.3% of DNA sequences are found in this file. Please note that Omega belongs to the alpha class of the four.

Table 1300 lists several independent conformations (i.e., 106.3, 106.107. 3.107.10B, 3.108.1o9.3.109) It shows the array of expected numbers in each class for Lee. 106 independent In conformations (ITs) expect to see 56 percent of the six omega classes. There are, but only 0.1% of the six alpha classes. Look The majority of sequences sampled come from classes for which less than 10 percent were sampled. . For example, peptides from the two phi, two omega, two alpha classes is isolated by splitting the library to bind to the target. Please think about it. What about the peptides associated with the isolated sequence? Think about how much knowledge you have.

Only 4% of the two Phi, two Omega, and two Alpha classes are San Amino acids from the Omega set are actually It is impossible to conclude that it is the best among them. There may be LEU in position 2 No, but ARG or SER might be better. omega six clans Even if you isolate a class of peptides, members of better classes will be added to your library. There's a chance to get attention that you didn't have.

I have seen several classes fully sampled in the 1071Ts library. be. However, the Alpha 6 class was only 1.1% sampled. Not pulled. 7.6・107ITs, 50% of the total amino acid sequence I am looking forward to the emergence of new trends. However, the alpha of three or more amino acids Classes that have assets are not sampled very often. (N　N　 K) To complete the sampling of 6 libraries, approximately 3.10'l Ts, the largest table 131 reported so far is (NNT)' (NNG) 2 It shows the expected value of a library coded by . The expected value of abundance is Regardless of the order of the cordons, or by grouping unchanging cordons into groups. It has nothing to do with it. The library is coated with 0.133 times more amino acid sequences. However, there are only 0,0165 times as many DNA sequences. In this way , 5.0・10'lTs (i.e., required for (N N K)' (60 times less than doing. The results are that (NNT)6 is somewhat better and ( N　NG　G　)' is somewhat bad, but it's not that bad. control The factor is the ratio of DNA sequence to amino acid sequence.

Table 132 shows the #DNA sequences/# for the cordons NNK, NNT, and NNG. The ratio of AA sequences is shown. For NHK and NNG, in the Pf phage Gene III is indicated by the phrase "presumed to be a stop loss" Therefore, it is speculated that PBD is revealed as an important gene part. So It is not necessary in any case for important genes such as Not yet. If less important genes are used, the analysis method may be slightly modified. right. Sampling NNT and NNG would be somewhat less effective. cormorant. Amino acid sequence in which (N　NT　　)6 is 3.6 times more than (N　N　　　　)5 Notice that it provides 1.7 times less DNA sequence and requires 1.7 times less DNA sequence. (N NT)7 provides twice as many amino acid sequences as (NNK)', but DN Notice that the A array is 3.3 times fewer.

In this way, simple mixing to obtain all 20 amino acids at special sites Although it is possible to utilize these simple mixtures of coated amino acids, The set becomes very distorted. This problem involves complexly modified cordons. can be used to solve the problem.

A complexly modified cordon Allows for all 20 amino acids and determines the abundance of the most preferred amino acid (mfaa). The maximum ratio of the minimally favored amino acid (lfaa) abundance to the ratio The nt distribution (rfxsJ) in the produced codon is based on the acidic moiety. As few stop codons as possible, even if there is an equivalent constraint of no acids , and for convenience, the third base is T or G, as shown in Table 10A. D encoding each type of amino acid, and the abundances indicated. Produces NA molecules. Other complex modified cordons include one or more can be obtained by relaxing the conformation.

This chemistry consists of a total of 20 amino acids, with acidic and base amino acids being equivalent. the most preferred amino acid (serine) coats the least preferred amino acid It is coded just 2454 times, the same frequency as acid (tryptophan). " “xsJ vg cordon is NNK or NNS offering only one cordon. Among the provided amino acids [F, Y, C, WSH, Q, 1, M%N, N, D, E] Maximally improves sampling for peptides that have some of the the The benefits of sampling are most pronounced when libraries are relatively small. becomes. Overlooking the condition of acidity and base equivalence and underestimating the stop cordon The results are shown in Table 10B9. The advantage of NNT cordon is that this patent registration It is being discussed everywhere. Non-optimized NNT is only 16 DNA sequences It provides 15 amino acids coated by As shown in Table 10C It is possible to improve the NNT with the distribution 1, which is almost the same amount as the 5 Provides amino acids (SER, LEU, HIS, VAL, ASP). 8 more three amino acids (PHE, TYRlILE, ASN, PROlALA, ARG.

GLY) is present at 78% SER abundance.

THR and CYS persist at half the abundance of SER. disulfide bond miku If we change the DNA for the protein, we will reduce the prevalence of CYS. is desirable. This distribution allows for the 13 amino acids found at high levels and No. Optimized fxs distribution has only 11 amino acids with high prevalence is allowed.

NNG cordons may also be voice optimized. Table 10D is nearly optimized (rALA J indicates the NNG cordon (approximately equal to [ARG]). Under this change, Four equally most preferred amino acids: LEU, ARG%ALA, and GLU There is. Please note that there is one acid and one base in this set. Sai. There are two equally least preferred amino acids: TRP and MET. 1f The ratio of na(!:mfaa is 0.5258. This cordon is repeated 6 times. peptide made entirely of TRP and MET was the most preferred amino acid. It has 2% commonality with the whole made peptide. Optimize this "The prevalence of (TRP/MET)6 in NNG vgDNA" I have to.

When synthesizing vgDNA using the "dirty bottle" method, only a limited number of mixtures are required. It is sometimes desirable to use it. One very useful mixture is “Optimized N It is called NS mixed. Among them, the first two positions of fxs mixture T1=0.24, C+=0.17, A,=0.33, GI=0.26 are averaged, The second position is the same as the first position, C3=G3=0.

It is 5. This distribution shows that amino acids of 5% or more ARG, SER%LEU , GLYSVALLlTHR, ASN, and LYS, and more than 4% Provides the above amino acids ALA, ASP, GLU, ILE, MET and TYR There is.

Cordons that have been modified to become more complex are interesting. this code is the same as the optimized NNT codon for the first two positions and The third position has TAG::90:10. This cordon is 5.5 pa Amino acids of 1 cent or more (ALA, ILE, ARG, SER, ASP, LEU, VAL, PHESASN, GLYSPRO, TYRl and Hls). 4 ・3% THR and 3.9% CYS are NNK (3,12 5 percent) are even more common than LFAAs. The remaining five amino acids are It is present in less than 1%. This cordon contains all amino acids Sequences with two or more low abundance amino acids are rare. When isolating SBD using this cordon, the first 13 It is theoretically certain that amino acids are tested at each position. optimized Similar cordons based on NNG can be utilized. .

Table 10E shows some characteristics of the optimized NNS (or NHK) cordon. It shows gender. The three equally most preferred amino acids AERG, LEU, Please note that there is a SER. 12 Equally Least Favored Aminos Acid, PHE, rLE, MET, TYR, HI S, GLN, ASN, LYSS ASP, GLU, CYS and TRP also exist. Five amino acids (PRO, THR , ALSlVALSGLY) are in between. The six repetitions of NNS are [A RG, LEU, and SER] and is only about 0.1% in phase with the array consisting of Amino acids [PHE, ILE, MET, TYR, Hls, GLN, ASN, LYS , ASPSGLU, CYS, SositeTRP]. this is , the optimized NNG' universal layer of (TRP/MET)6 in vgDNA. Not only is it nearly 20 times lower, this lower generality applies to 12 amino acids.

Diffusion induced Diffusion triggering can be applied to any part of the protein and in any case However, it is most appropriate when the substance to be bound to the target is set. Expansion DN with a small amount of one or more other activated nts A synthesis (i.e., nt phosphoramidite-nt-phosphoram idites) for each activated pure nts (disabled This can be accomplished by Preferably, the level of the spikes is A small portion of a product (for example, 1% of 0.0001%) Contains the initial DNA sequence (in the amount of leprosy; this includes single, double, triple , and ensures the occurrence of further triggers, but recovery of the underlying sequence is This is a possible outcome.

111, D, associated with changes in microproteins with significant cysteine Special consideration Some desirable simple or complex modified cordons can be encodes a set of amino acids containing This is because several encoded binding domains , one or more cysteines in addition to the invariant disulfide-bonded cysteine It means that it is characterized by For example, each NNT coded position In this case, there is a possibility that cysteine can be obtained in the internal rotation of 16 chances. six pieces If the -don is replaced, the fractional value of the domain containing the additional cysteine is 0.3. It is 3. Odd numbers of cysteines cause complications. Perry and f See etzel (PERR84). On the other hand, it contains a large number of disulfides. proteins that contain disulfides, i.e., cysteine that does not form trypsin. Contains The possibility of unpaired cysteines is expressed in several ways. can be handled. That is, First, the altered phage population was created using SulfoLink. (Pierce Chemical Company, Rockford, l1 Catalog number from linois, 61105 is 44895 H) Immobile reagents that tightly bind thiols may be ignored. Pie Another product from rce is TNB-Thiol Agarose (Cat. The program number is 20409H). BioRad uses Affi-Get for this purpose. 401 (catalog number 153-4599).

Second, changes that exclude cysteine can be used, such as those described below.

That is, NHT--[F, S, YlL, P, Hl 1, T, NSV, A.

D] provide VNS-Ikuchino, P2, H% Q, RJ, LM, T2, N, K.

S, V2, A2, E%D, G2] - [L”, S, W, P, W ,R',M,T,to,R.

5NT--[L, P, H, R, V, A, D, G] - provides R, VSA, E, G] to [M, T, RMG--[T, to, A, E] VNT--[L, P, H, R, I, T SN, S, V, A, D, G] or RR3--provides [N, S, R, D, E, G2].

However, each of these schemes has one or more advantages compared to NNT. It has more disadvantages than that. i.e. a) fewer amino acids are tolerated; b) Amino acids are not provided on average; C) Acidic and base amino acids cannot possibly be equivalent, or d) a stop codon occurs. death However, NNG, NHT, and VNT are just as useful as NNT. NNG encodes 13 different amino acids and one stop signal. only two The amino acids appear twice in the 16 mixtures.

Third, it can enrich populations that bind to predetermined targets; and for the extra cysteine, the selected sequence (post hoc-1) Immediate causality) can be evaluated. The cysteine provided to constrain the conformation. Those containing a large number of cysteines are completely useful. other than designed disulfide linkages may occur. Based on the isolated DNA sequence This is not to say that binding domains defined as such are not appropriate in any case.

The suitability of isolated domains for chemical and biological synthesis of chemically synthesized peptides Best determined by chemical evaluation.

Finally, use a solution such as Ellman's reagent, iodoacetate, or methyl iodide. Free thiols can be blocked with a reagent solution. The test solution is especially free thiol. binds to molecules and does not interact with disulfides, and the adjusted fluors in the population. Leave the arge alone. Drugs that block microprotein binding You should be aware that there is a possibility that you may change your characteristics. .

In this way, we hope that different binding domains may be discovered. A variety of drugs may be used to block the disease. Blocks thiols The altered population of genetic packages is fractionated for conjugation. isolated If the DNA sequence of the bound microprotein contains an odd number of cysteines, the combined The method of construction is to make each bond with an odd number of thiols properly blocked. It is utilized to provide micro-proteins. Nisoute (Nl 5H86, Nl5H86, and the papers cited here), each thiol is Multiple cysteines protected by different types of blocking groups 1, these groups disclose methods of peptide synthesis, including selective disulfide pairing can be controlled. one thio Either the rule remains blocked or it is unblocked and a different attempt is made. We are considering using a modified scheme in which the liquid is blocked again.

111, E. Planning of the Second and Subsequent Change Rounds The method according to this invention is , the attachment of a binding domain that has high affinity for a predetermined target. Allows effective collection of information regarding amino acid sequences. Single round changes or affinities Even if very useful binding domains were obtained from enrichment, the highest possible affinity We believe that many rounds are necessary to win the character and uniqueness.

Even if you make some kind of bond with the target in the first round of change, the target The affinity for get is still too low, and further improvement will be achieved by changes in 5BDs. It will be. Desirably, the process is progressive. That is, for each change A cycle produces more for the next cycle of change than the previous cycle produced. Create a good starting point. Set the level of change so that ppbc+ and a large number of sequences related to ppbct sequences are present in detectable amounts and This ensures a step-by-step process.

If the level of variation is so high and the ppbd sequence present at such a low level Therefore, there is no discernible opportunity to say that there are no transformants that manifest PPBD. Also, there is a possibility that the highest SBD in the next round will be worse than PPBD. be.

At unnecessarily high change levels, each round of triggering is independent of the previous round. There is no guarantee of progress. This method leads to valuable binding proteins. There is a possibility that 1 However, repeating experiments at this level of change yields progressive results. does not bring about Excessive variation is not desirable; It is not a property of nothingness. Most of the data from the previous change cycle is retained. When a large number of different surfaces related to the PPBD surface are produced, the process is progressive.

If the level of change in the previous change cycle was chosen correctly, the change was made correctly. The amino acids selected to be present in the residue group are the ones that are best determined.

As the environment changes for other residual groups, it is appropriate to change them again. at the same time There are many more interesting residue groups than can be changed, so there are many more interesting residues than can be changed. one that has never been used (highest advantage) or one or more Will we continue to select any remaining groups that could not be changed for Ikuru? I don't know.

The use of NNT or NNG modified codons is a modified library. This results in a very effective sampling of The reason is (different amino acids acid sequence) and (different DNA sequence) ratio than that for NHK, or is much more unity than that for the optimized yg codon (f xs) Because it is close to. In any case, several amino acids are omitted in each case.

Both NNT and NNG contain important classes of amino acids (hydrophobic, hydrophilic, acidic, Choose a binding domain that tolerates members of different types (base, neutral hydrophobic, small, large). After choosing, subsequent changes and selections achieve a high degree of affinity or specificity. During this second change, it would be desirable to The possibility of amino acids will be considered.

I hope some examples will be helpful. Suppose you obtained PRO using NNT. Let's decide. This amino acid can be used as either NNT or NNG. PRO is set [PRO, LEU.

VAL, THR, ALASARGSGLYlPHE, TYR.

With the best amino acids from CYS, Hls, ILE, ASN%ASP, SER It is theoretically certain that there is. [PROlTRP, GLN, MET, L Let's try a set that includes [YS, GLU] next. allowed by NNG This set is the desired set.

What would happen if you obtained HIS instead of the target substance? It can combine with the sol ring and also form hydrogen bonds from the imidazole ring. can. Tryptophan is hydrophobic and aromatic, and is Can bestow hydrogen on xceptors and is also excluded by NNT cordon . Methionine is also excluded and is hydrophobic. .

Thus, one desirable course is [HIS, GLN.

ASN, LYS, TYR, CYS, TRP, ARG, SER.

By using a modified cordon HDS that allows One, If the first round of change is completely unsuccessful, a different pattern of change should be used. For example, one or more interactions are defined within a domain. If possible, the changed residues in the next round of changes will be changed within the first change. Must be from a set other than the committed set. Failures will occur repeatedly In such cases, it is possible to change to a different IPBD.

IV.Display strategy: Displaying the heterologous binding domain on the surface of the “gene package” IV, A. General Requirements for Genetic Packages A number of different but related In order to obtain the manifestation of a potential binding domain with The goal is to generate a heterogeneous package of genetic packages. This genetic package each coats a potential binding domain for the target of interest. a second DNA sequence that coats the first DNA sequence and a second DNA sequence that coats the 1. In this way, the proteins on the outer surface Although natural to the genetic package, the potential binding domain (or the parent binding domain with which it is associated) . The potential binding domain is the chimeric protein equivalent (or It is the basis of a genetic package that displays on its outer surface a processed form of

The members of the population that embody the desired bonding properties are very small, e.g. It may be less than 10r'.

Once the members of this population are separated from the unbound members, they are amplified. It must be possible to Cultivating cells with variable genetic material It is the most powerfully amplified and desirable one. genetic message is amplified in vitro, ie, by PCR. However, this is the most Not a desirable method.

Preferably, GP is as follows. In other words, ■) potential 2) a genetically modified theoretical mechanism encoding a binding domain; 3) those that interact with the target substance during affinity separation; engineered to reveal binding protein domains with the potential to and 4) affinities are separated but retrievably manifested. Those that have genetic information that encodes a synthetic domain. Preferably, the GP It remains variable even after affinity separation. Desirable GPs are nutritional bacteria. bacteria, bacterial spores, and especially bacterial DNA viruses. eukaryotic Biological (eukarynte) viruses can be used as genetic packages right. But they are not desirable. The genetic package is bacteria If it is a cell or if it is a veliplasmically assembled phage , the means of manifestation has two elements. The first element is the first expressed substance in the cell ( If Banken is a phage, it is a secretion signal that leads to the inner capsule of the host cell.

This secretory signal releases the processed, mature, and potential binding protein. cleaved by a signal peptide to produce The second element is the package The outer surface transport system leads the protein to collect processed proteins on its outer surface. It's Gunar. Desirably, this outer surface transport signal is native to the genetic package. It is derived from surface proteins found in

For example, in a desirable experimental example, a hybrid gene has one signal sequence. sequence (i.e., the signal sequence of the bacterial phoA or bla gene, if is operably linked to the M13 phage gene I11 signal sequence). or coat proteins of filamentous phages (i.e. M2S) (i.e. D encoding M13 gene III or gene VIII protein) NAI: Encodes an operably linked potential binding domain. It is made up of DNA. The expressed substance is the endothelial membrane (lipid double membrane) of the host cell. ). Therefore, the signal peptide is a processed hybrid protein. It is divided to leave the group. The coat protein of this hybrid protein The C-terminus of such an element becomes trapped in the rebid bilayer. hence the hive Lid proteins do not escape from the veliplasm. (This is a wild typeco This is a typical protein. ) single strand of unfinished phage particles The DNA enters the periplasm, which is the coat tongue of the wild type. Proteins and hybrid proteins are collected from the lipid membrane. hybrida Proteins thus have potential binding domains exposed on their outer surface. leaves the cell and becomes incorporated into the surface coating of filamentous phages. (In this way, the host In this example, the filamentous cells, not the bacterial cells, are It is "Nken". ) A secretion signal is required for the display of the potential-binding domain. If necessary, in particularly desirable experimental cases, the hybrid gene may be expressed. The bacterial cells revealed are of the "secretion permissive" lineage.

The genetic package is assembled inside the bacterial spore or coat within the cell. If it is a phage (such as PhiX174 or Lambda), the host bacteria Secretory signals directing the expressed substances to the endothelial membrane of Telia cells are not required. this In some cases, the means of expression is simply an external surface carrying signal, typically a spore or It is a derivative of phage coat protein. .

Desired OSPs for several GPs are shown in Table 2. This section For the melting of the osp-ipbd in the PBD and OSP-SBD melting (should also apply).

Use of phages as IV, B, GPs - IPBD is a disulfide-bonded microorganism If it is a protein, IPBDs do not fold into the cell (this After the phage is released from the cell, these proteins are ), periplasmically assembled fur It is desirable that IPBD is large or cannot be dissolved in formulations. In cases requiring a mouth (such as the Fe4S4 group), intracellularly assembled Phages are preferred. The reason is that the compounding mouth is Be1noplasm. This is because IPBD may not be folded when secreted because it lacks . When changes are introduced, multiple contaminants with different PBDs on their surfaces carries the gene for one PBD with at least several copies) /% IBRIT GPs can be generated. It is a low L1 large number of contamination ( This is possible due to contaminating cells carrying phage under conditions that resulted in MOI'). The ability to keep sex to a minimum (desirable).

Since there is little enzymatic activity associated with intact mature phages, showing mature phage particles that are metabolically inactive; Bacteriophages are good candidates because they are inactive in the host cell.

For a given bacteriophage, the desired O12 is usually This is one of the phages present on the surface of P. phage. Despite this, M2S g O12 like ill tannoplasm (5 copies per phage) It may be a good choice as O12, which is responsible for the manifestation of PBD.

It is desirable that the wild-type 6sp gene be conserved. 1pbd remains The gene fragment is either inserted into the second copy of the receptor osp gene or de novo Possibly inserted into the engineered osp gene. The asp-ipbd gene is It is desirable to be placed under the control of a well-organized promoter.

To insert a fragment of the 1pbd gene, the user inserts a fragment into the candidate O8P gene. (1°) The coat of the majority of bacteriophages is It is regulated. In such bacteriophages, virus particles The residual group of the parent O8P that interacts with other proteins in (virion), It is important to keep the 05P-IPBD melt in operation. M2 For S glII, the last 100 residues (BASS90) (or It may be sufficient to keep M For 2S gVTII, it is desirable to retain all mature protein. 7 such truncated gillllll proteins all proteins are 7 Alongside the complete gllll protein, as required for 7-ci contamination. It will be expressed.

Includes Ml3, fl, fd, Ifl, Ike, Xf, Pfl, and Pf3. Of particular interest are filamentous phages. The main coat proteins are It is encoded by gene III. 50 amino acid maturation gene Vllll code The protein is synthesized as a 73 amino acid precoat (ITOK79). Ru. The 123rd amino acid is responsible for the insertion of the unfinished polypeptide into the cell endothelial membrane. It constitutes a typical single array.

One E, Co11 signal peptide (SP-1) has amino acids 18.21, 23 and, to a somewhat lesser extent, residual groups 22 and prico residue groups 23 and 24 of Approved cuts between. After removing the signal sequence, the mature code The amino terminus of the protein is located on the veriplasmic side of the endothelial membrane. carboxy-terminated is present on the cytoplasmic side. Approximately 300 of the mature 50 amino acid coat proteins 0 copies participate in parallel in the endothelial membrane.

The sequence of gene Vlll is known, and the amino acid sequence is.

utilizing the 1acUV5 promoter and in conjunction with the Lacl' inhibitor. can be coated into synthetic genes. IacUV5 promoter is induced by IPTG 1, the mature gene III protein forms a coat around the circular 5sDNA. to be accomplished. The three-dimensional structure of r1 virus particles is known with moderate resolution. Legacy The amino terminus of the gene Vllll protein is located on the surface of the virus particle, and Because of this, it is a desirable connection site for potential binding domains. Gene Vl Several adjustments were made to ll and are described below. M1 The two-dimensional structure of the three coats is implicit in the three-dimensional structure. Mature M13 gene V The lll protein has only one domain.

A three-point gene consisting of the following was constructed. Namely, 1) the secretory part through the endothelial membrane; DNA coating the signal sequence leading to components (2) and (3).

2) DNA encoding the mature BPTI sequence, and 3) mature Ml3 DNA coating gVIII This gene is active on the surface of M13 phage. It causes BPT I to appear in a powerful form.

The amino acid sequence of M13 precoat (SCHA78) is called AA-seql, It is as follows. That is, MWVIVGAT Engineering G Engineering KLFKKFT5iK The best site to insert a new protein domain into ASM13CP is indicated by the arrow. As shown, it is after A23. The reason is that 5P-1 pre-empts after A23. This is because it cleaves the coated protein. Proteins that can be secreted are It appears linked to mature Ml3CP at the amino terminus of. mature M13C Since the amino terminus of P is located on the outer surface of the virus particle, the introduced domain The in will be displayed on the outside of the virus particle. M13CP becomes qualified 2M membrane The fact that the mechanism by which it appears has not yet been elucidated means that the direct gene Vl of bpLi It is possible that insertions into ll may not produce a functional fused protein. to raise. In addition to the melting signal sequence, for example, the phoA sequence (MKQST IALALLPLLFTPVTK A,,,,,,,,) OIAR91) It may be necessary to change to.

Mtlrks et al. (86 in MAR) showed that the phoA signal peptide is mature B We showed that PTI can be directed into the E. col i periplasm.

I. Another way to manifest PBD is to use genes that contain some or all of the gene III. 9, this gene is expressed as a chimeric gene domain containing The offspring coat one of the minor coat proteins of Ml3, genes Vl, V ll, t■X also coat minor coat proteins. these minors Each of the proteins has approximately five copies per complete mature virus particle. exists within. and they are related to morphogenesis or contamination. to this Comparing the major coat protein to a complete mature virus particle Exists in more than 2,500 copies of each. Genes Vl, VI I, and IXta The protein is present at the end of the ripe virus particle.

These three proteins are not metastatically manipulated. (RASC86).

A single strand of circular phage DNA contains approximately five genes of gene II DNA is trapped in a helical coat of protein. extrusion through a batch of coat proteins associated with the membrane in such a way that It will be done. (WEB378). DNA is not based on pairs (it is a virus) Tight rigor on the genome imposes restrictions). Rather, the base is independent of the array and insert them into each other.

S+ith (SMIT85) and delacruz et al. (DELA88) Insertion into II This indicates that it is the cause of expression on the surface. The mini protein gene is 5w 1th and de Gene 11L of the site used by IaCruz et al. corresponds to the main border or to the surface loops of the protein. gene III in one codon, or the amino terminus of the mature protein. It may be fused to gene III at the end.

In the experiments described in all references, the fd of a single regulated gene III was The containing vector is used. In this way, all five components of gill P is adjusted in the same way. Gene III is quite large (12?2b, p, or about 20% of the phage genome), and the entire gene It is not certain whether replication is stably inserted into phages.

Furthermore, all five copies of the glII protein are present in the fully mature virus particle. exists at one end of the A divalent target molecule (such as an antibody) is the resulting complexity is irreversible. It would be possible. If the GP bound to the target cannot be reversed, the target The gene encoding the novel polypeptide that has the greatest affinity for the target. It significantly interferes with the affinity isotope enrichment of GPs that carry gene sequences.

In order to reduce the possibility of irreversible complications occurring, the carboxy A second synthetic gene encoding the terminal portion may be available.

The carboxy-terminal portion of the gene III protein is incorporated into the phage. This may cause For example, (arginine identified by cordon 398) The last 29 residues are responsible for the incorporation of the fused protein into the phage. It may be enough.

Alternatively, the mature girl protein, i.e. the last 15 of gene II It may contain a final spore domain of amino acids 0 to 160. parable For example, one gene consisting of the following (from 5 feet to 3 feet) can be manipulated. May. That is, ■) promoter (preferably regulated), 2) ribosome binding site, 3) Initiation cordon, 4) Secretion f7) Functional signal guiding parts (5) and (6) through the endothelial membrane Naru peptide, 5) DNA coating IPBD.

6) DNA encoding residue groups 275 to 424 of M13 protein, 7) Conversion stop cordon, and 8) (Optional) Transcription stop signal.

Since the wild type gene III is left, the unconverted gene I II will exist. Alternatively, gene VIII protein as osP may be utilized and may control osp-i pbd melting. Therefore, one or several copies of the fused protein are expressed on the phage.

3. M13 genes VI, Vll, and IX proteins are not manipulated after conversion. child The route by which these proteins are incorporated into phages has not yet been reported. stomach. These proteins are required for normal morphogenesis and phage infectivity. It is.

These molecules (gene Vl protein, gene VII protein, and gene IX protein) from a) the cytoplasm, b) the periplasm, or C) lipids. It is unknown whether the phage is connected to the phage from inside the double membrane.1. These proteins can be used to introduce IPBD onto the phage surface using one of the following methods: You can take advantage of one of these qualities. That is, 1) ipbd::pmcp.

2) pmcp:: 1 pbd.

3) Signal: : 1pbd : : pmcp, and 4) Signal pm cp: : 1 pbd.

So 1pbd coats the expression for the first potential binding domain. pmcp is the phage minor coat protein V. i　VI　Displays the DNA coating for one of I and IX, The signal is the phoA signal (MKQST I ALALLPLLFTPVT indicates a functional secretion signal peptide such as KA), and “~:” , displaying gene fusion in the frame. Displayed melting is caused by known promoters, desired Preferably a regulated promotion such as IEICUV5, tac, or trp. placed downstream of the data. Melting (1) and (2) show that minor coat proteins are when attached to the phage from the cytoplasm or by autonomous insertion into the lipid bilayer. It is appropriate to connect the Melting (1) is a minor coat protein Melting (2) is suitable when the carboxy terminus is free; Appropriate if they are separated. Melting (3) and (4) are minor coat proteins If the phage connects to the phage from the periplasm or from inside the lipid bilayer membrane. Suitable for connecting to phages. Melting (3) is minor coat protein Melting (4) is suitable when the carboxy terminal is free. Appropriate if there are

Similar structures can be constructed with other filamentous phages. PF3 is IncP-1 plastic Pseudomonas aerugenosa (Pseudomr+n) harboring Sumido It is a well-known filamentous phage that infects S. aerugenosa cells. . The major coat protein of PF3 contains a signal peptide that guides its secretion. It is normal not to have one. Is that sequence the residual group ASP? , A RG 37, L Y S 4. , and charge PHE 44-COO. the array is exposed Consistent with the amino terminus. In this way, IPBDT is applied to the surface of PF3. To effect expression, a three-point gene was constructed consisting of: That is, 1) Pseudomonas aerugenosa (IPBD portion) melted in (3) within the frame (preferably known as causing secretion) A known signal sequence, 2) inside the frame (3), IPBD is A single gene fragment coded for 3) DNA that coats the mature Pf3 coat protein.

Optionally encodes a flexible chain of amino acids from 1 to 10. DNA or one special protease (i.e. factor Xa). The amino acids forming the recognition site are the ipbd gene fragment and the Pf3 coat protein. It is introduced between protein genes. This three-point gene is introduced into PF3. , does not interfere with the expression of other Pf3 genes. To reduce the possibility of genetic modification Therefore, the third part has a large number of silent mutations relative to the wild-type gene. It is well designed. Even if the signal sequence is split, the IPBD remains in the replasm, and the mature coat protein also acts as an anchor. Acts as a phage assembly signal. This molten protein is the wild type lipid bilayer by a route other than that allowed by coat proteins. It's not a problem to rest inside.

Bacteriophage Phi X as described in W090102809 Phages such as 174, large DNA phages such as lambda or T4, etc. Even RNA phages can be used as GPs with proper adaptation and adjustment. May.

IV, C, Bacterial cells as genetic packages The following well-characterized You may choose one of the bacterial strains. That is, ■) those grown in culture; , 2) engineered to display PBDs on its surface, and 3) affinity What is selection and mutuality?

The desired genetic package within the bacterial cell is Salonella typhii+urius, Bacillus 5ubtilis%Pseu doaonas aeruginosalIVibrio choleae, Klebsielln pneumonialNeisserta gonor rhoese, Ne1sseria meningitidis, Bocte roides nndosus.

Moraxella bovis and especially Escyerichia coli It is. Potentially binding miniproteins bind to the outer surface of chimeric bacteria ( It may be expressed as an insert into the cytoplasm (O3P). All bacteria are that They display proteins on their outer surfaces. E. coli is a desirable bacterial GP , and for that reason fSLamB is the desired osp.

The majority of bacterial proteins reside in the cytoplasm, but others reside in the periplasm. in the plasma membrane (located between the plasma membrane and the cell wall of Gram-minus bacteria). transported or delivered to or fixed on the outer surface of the cell. Furthermore, that Other bacterial proteins are exported (secreted) into the media surrounding the cell. ). Proteins recognized by cells and exported out of the cytoplasm The characteristics of the proteins that cause the protein to be expressed on the cell surface are These signals will be referred to as "surface-transported signals." Gram-minus bacteria is 03P They have outer membrane protein 1K (OMP), which forms a subset of s. Many OMPs have a span that is one or more times the length of the film. OMPs as outer coating Localizing signals are encoded within the amino acid sequence of the mature protein . Bacterial coat proteins contain something called a signal peptide. It is initially expressed in a containing precursor form. Precursor proteins are transported to the endothelial membrane , and the signal peptide moiety is pushed into the field of the belliplasm. Served. There, it is cleaved by a "signal peptidase" and then The remaining "mature" proteins can then enter the vellastrism.

Once other intracellular mechanisms recognize the structure in the mature protein and adapt it, Indicates that the desired area is in the outer membrane, and then transports it to that area. .

Leader or signal from one protein coded for peptide The DNA of protein X is combined with other proteins, protein The expression of the chimeric gene is connected to the DNA sequence encoding the protein. It is well known that protein X is the cause of free expression in the periplasm. Are known. Utilization of export permission for bacterial strains (LISS85.5TAD89) increases the probability that signal sequence melting will direct the desired protein to the cell surface. .

08P-I PBD fusion protein has a highly unregulated outer membrane part Therefore, it is necessary to play a structuring role in the outer coat of Gram-minor bacteria. No 3. One or more sites exist for large OS Ps. Dew. Then the asp is discontinued on that site and melted to 1 pbd. In this case, cells expressing lysis will reveal IPBDs on the cell surface. omp The fusion of a gene fragment and one X gene fragment causes up to the X expressed in the outer membrane. I'll lead you. (CHAR88b and c, BENS84, CLEM81). Like that If a melt is formed, substitute ipbd for X in the DNA sequence. One osp-ipbd can be designed by Otherwise If successful OMP-IPBD melting occurs while expressing the melted gene, The best am Desirably sought by melting the fragments of p into one pbd Ru. between omp and 1pbd to maximize the chance that IPBD will be exposed. Use on OMF to take a melting point or points Use available data. (Spacer DN coated with a flexible linker) A facilitates disclosure (linker consists of GLY, 5ER1 and ASN) can be placed between fragments derived from osp and fpbd to

Alternatively, you can abort the OSP in several sites, or create a variable length ASP break. The OSp fragment is melted to 1 pbd and the melt is expressed. Cells are screened or selected, and IPBDs are revealed on the surface of the cells. do. , Freudl et al. (FREU89) reported that O8Ps (O mpA) fragments were shown to be incorporated into the tack membrane. one more An alternative method is to use short sections of random DNA in the melting of amp fragments and ipbd. ment and displaying a phenotype of IPBD manifestation. Screen or select the resulting altered population for bars .

In E. coli, the LamB protein is a well-understood O12; can be used. E. coli LamB protein is S. typhimur ium, V, chnlerae, toni, pneumonia in a functional form Since it is expressed, this as one melt to E, coli L a m B Both species and genera can exhibit populations of PBDs. K, pneu monia contains maltoporin (ma I tnpnrin) similar to LamB. (WEHM89), which can also be used. P, aerugi For nosn, one homologue of the DI membrane protein LamB) is available. (T RIA88).

LamB extends to the outer coat if a functional N-terminal sequence is present. be transported. In addition, the first 49 amino acids of the mature sequence lead to successful delivery. required for.

(BENS84). Regarding other 03Ps, LamB of E, col i is Synthesized with a typical signal sequence that is later removed. LamB protein and other The homology between parts of the outer membrane protein OmpC1OmpF and PhoE is Including the homology between the B amino acids (39 to 49) and the sequence of the Ike protein, It's being served. (NIKA84). These subarrays are responsible for the proteins carried in the outer membrane. You might even be called a prankster.

The amino acid sequence of LamB is known. (CLEM81). And one model Dell has also developed how it fixes itself to other membranes. It is. (Including others, investigated by BENZ88b.) Mordos (maltose) and the site of the phage binding domain are also known. (HEIN88 ). This information is used to provide a chimeric O12 that is displayed on the outer membrane of the bacteria. Several strategies can be achieved by inserting a PBD that may be incorporated into LamB. may be identified.

PBDs are expressed by chimeric trans-capsule proteins such as LamB. If necessary, the PBD is inserted into loops normally found on the surface of cells. obtain. (Comparison of BECK83 and MANO86). Alternatively, one 5 fee The O8p gene of the tosegment may be fused into the ipbd gene fragment.

As the melting site, select a location corresponding to the loop exposed to the surface of O12, and 2 carboxy-terminal proteins are excluded. In LamH, up to 60 amino acids with the resulting manifestation of a variant epitope. It may have been inserted. (CHAR88b and C). OmpC, OmpA , OmpE, and PhoE, since their structural features are very similar, these proteins I expect a similar function from .

Although LamB may be characterized as a binding protein, the present invention Please note that the inside part is used to provide the OSTS. Please. Its binding domain is unchanged.

Outside of other bacteria such as OmpA, OmpClOmpF, PboE and pilin Surface proteins may be utilized in the case of LOmB and its homologues. O mpA because it is very abundant and its homologs are widespread. Gram-minus bacteria are known among the genus and are therefore of special interest. It is something that Baker et al. (BAKE87) reported that the outer coat of E. coli investigated protein incorporation in and residue groups 19-32.62-73.10 5-118, and 147-158 are exposed on the cell surface. 　mp　A topological model is cited. (VOGE86). approx. cordon One 1pb encoding fragment at 111 or approximately at cordon 152 The insertion of d appears to be responsible for the appearance of IPBD on the cell surface. Om Regarding pA, see also MACI88 and MANO88. Pseudomonas Porin protein F of A. aeruginosa was cloned and OmpA of E. coli has sequence homology. (DUCH88). This homology is proline Although not sufficient to allow prediction of residual groups exposed on the surface of protein F, The method used to determine the topological model of OmpA has been applied to porin protein F. may also be used. Regarding using OmpA like one O12 Related studies are included in BECK80 and MACI88.

Misra and Ben5on (illsR88a, formerly 5R88b) have a residual group G L　Y　184 and LEU2SQ are exposed on the cell surface, as well as others. , we disclose the topological model of E, cnli Om p C, which predicts that ing. In this way, approximately the codon of E. coli o mp C cells is at 164, or approximately at 250 of the cordon, or S, thyph One 1 pbd at the same codon of imurium o m p C gene Insertion of gene fragments may cause IPBD to be expressed on the cell surface . The ompC genes of other bacterial species may be used. To OmpC Related studies include CATR87 and CLIC88.

E, cnli OmpF equals one highly abundant □SP, 10' copy or large cells. Pages et al. (PAGE90) exposed seven surfaces. announced an OmpF model that displays the segment. one ip Melting of the bd gene fragment can be done as a single insertion or as a 3 foot section of ompF. One of the displayed areas also has a functional ompF. bd gene may be produced. And its expression is caused by IPBD on the surface of the cell. It guides us to manifest. In particular, about cordon 111.177.217, if Melting at 245 must lead to a functional ompF::i pbd gene. not present. Regarding OmpF, REID88b, PAGE88, BENS88 ．． See also 70MM82 and SODE85.

Billy proteins are found in these pilated cells. Because it expresses multiple copies of the protein, it can also be used in other species (N, gono rrhoeae, P. aeruginosa, Moraxella bov is.

Bacteroides, nndosusl and E. coli) are related This is of particular interest because it expresses Getznff and his collaborators ( GETZ88, PAGE87, SOME85) let, Neisseria gonorrhoeae Teruo structure and its gonococcal fiber protein are similar to tobacco mosaic virus. Rus protein forms four helical bundles with a mechanism similar to myohemerythrin It is predicted that it will happen. In this model, both the amino and carboxy terminations are exposed. The amino terminus is methylated. EI Iew+an(ELL E88) investigated pilin in Bacteroidesnodosus and other species. , the difference in serotype may be related to the difference in pilin protein, and the most It states that the major upheaval occurs in the C-terminal region. Amine terminus of pilin protein The edges are highly preserved. Jenn ings et al. (JENN89) A fragment of the virus Aphtosa (residues 144-159) was injected into the N. pylori B, nodosus type 4 fimbriae protein, which is highly homologous to the protein. P. Expression of 3-foot terminal melts in S. aerubinosa led to biological fungal species. They found that the protein produced detectable amounts of molten protein. . Jennings et al. did not alter the heterologous epitope. What about them again? They did not suggest any variation between the last pirin residue and the [flexible phosphorus]. one GLY-G between the first residue of the heterologous epitope and the LY linker was inserted. In this way, the desired location for bonding the IPBD is was found to be carboxy-terminated. Tested by Jennings et al. The special internal melting (JENN89) caused by P. aeruginosa Although it seemed fatal, the exposed loop of the bundle could also be exploited right. Regarding pilin, see also E85 and 0RND85 in MC.

Judd (JIJDD86, JUDD85) is N, gonorrhoene studied protein IA and found that the amino terminus was exposed.

In this way, a means of revealing the I PBD on the surface of the N, go orrhoeae. Attach the IPBD to or near the amino terminus of the mature P, IA as Can be done.

One model for the PhoE phase of E, col i is van del Ley et al. (VAND86). This model has eight loops exposed. It warns you that something is about to happen. Insertion of IPBD into one of these loops This seems to lead to the appearance of IPBD on the cell surface. Residue group 158.201.23 8, and 275 are the desired sites for insertion of the IPBD.

Other available 03Ps are E, which is a nutrient acceptor usually found in small amounts; coli BtuB, FepA, FHuA, IutA, FecA and FhuE (GUDM89). The genes for all these proteins have been sequenced. However, a topological model is not yet available. Gudi + unsdnttir et al. GUDM89) discloses certain residual groups on the BtuB surface, periplasmic. and to determine the functionality of viral Btub::FepA melting. We therefore began constructing such models for BtuB and FepA. Car Mel et al. (CARM90) reported a study of similar nature on FhuA. are doing. The entire genus Neisseria has already been identified1, and in many cases It expresses a cloned outer surface protein for iron transport. MOI ? See also S87 and MOR388. Many crumb-minus bacteria are Generates one or more phospholipases (ph-osphor 1 phase) It is appearing. E, col i Phosphate lipase A, the product of the pldA gene is de Cloned and sequenced by Geus et al. (DEGE84). They are Tampa We discovered that the protein is expressed on the cell surface without post-transferential processing. one 1p The bd gene fragment can be connected at the end or as a loop in the protein. It can be inserted at a site thought to be coding. The phospholipase A is arrives at the outer surface without removing the signal sequence, and its signal sequence is PldA: IPBD melting water is not allowed.

In this way, one PIdA::IPBD or IPBD::Pld A melt can be secreted into the periplasm by adding an appropriate signal sequence. This may cause the problem to occur. In this way, one 1pbd fragment is one Pbd fragment. In addition to having a simple binary melt at the end of the ldA, the following structures are tested: Should. That is, 1) ss::1pbd::pldA 2) SS:: pldA・1pbdPIdA::1PBD protein Once liberated in the periplasm, no one remembers how it got there. and the structural characteristics of PldA that resulted in it being located on other surfaces. The symptoms would lead to melting to the same target site.

IV, D, Bacterial spores as genetic packages Bacterial spores are GP candidates has desirable characteristics.

Spores are susceptible to chemical and physical agents such as vegetative bacterial cells or ) is more resistant than 7-di. And for this purpose, a variety of affinity selection states are available. Permit use. In addition, Bacillus spores actively reproduce. It does not degrade or change the proteins on its surface. bacillus spores, and More specifically, B, 5ubt i l is spores therefore It is a sporoidal G Ps. Details in W090102809 As explained in detail, the heterologous binding domain is B, 5ubtili s External surface tags such as those encoded by the cotC or cotD genes It may be introduced into proteins.

Typically, cells, spores, or viruses can be used as genetic packages. It is desirable that outer surface proteins have already been identified. I can be manipulated to reveal PBD. However, W090102809 As explained in , this invention is not limited to such genetic packages; , as external surface-carried signals may be generated by variation and selection techniques. .

V, E Consideration of gene structure and expression (i) The pbd-asp gene would be: i.e. a) fully synthetic b) a composite of natural and synthetic NA, or C) a composite of natural DNA fragments. . Importantly, the pbd segment, as mentioned above, is a member of the large and diverse PBD This means that it could easily be changed to encode the s family. Synthesized 1p bd segment allows it maximum control over the placement of restricted sites This is desirable because it has been

Supplements the region flanking the osp-ipbd gene flanking its 3 feet. As a supplement to the core molecule that exists as a The core molecule present in the sequence is required for array construction.

The sequences of the regulatory parts of genes are taken from the sequences of natural regulatory elements. sand That is, a) promoter, b) Shine-Dalgarno sequence, C) The transcriptional terminator-0 regulatory element is also known from the knowledge of sympathetic sequences in the natural regulatory region. can be designed. An array of these regulatory elements is connected to the region that performs the code. Ru. The regulatory site may also be inserted into a regulatory region that allows convenient adjustment. or adjacent to each other.

A key feature of affinity separation is that PBs that have a high affinity for the target GPs supporting Ds (derived from IPBD) for the target The goal is to separate PBDs, which have low affinity, from supporting GPs.

If the elution amount of GP is dependent on the number of PBDs on the GP surface, low affinity, One GP that supports a large number of PBDs with GP (PBD, ) is a highly parent One GP that supports a small number of PBDs with compatibility and PBDs (PBDZ) It may also elute with the Regulation of the osp-pbd gene is preferably carried out by Sufficient PB for the majority of packages to influence good separation according to affinity The goal is to make D obvious. Control the level of asp-pbd expression The use of regulatable promoters to closely control the characteristic behavior of the altered population Allows for adjustments to be made.

Engineered gene synthesis induction in vegetative bacterial cells is achieved by 1acUV5, tr A regulated promoter such as pP or tac (MANI82) has been used for a long time. The factors that regulate the amount of protein synthesized are It is well understood that a wide variety of heterogeneous proteins can be found in E, col i, B, 5ubti1is and other host cells (at least in moderate amounts). can be generated. (BET788). Preferably, the osp-ipbd gene The promoter for is subject to regulation by one small chemical inducer 1, for example, the lac promoter, and the hybrid trp-lac ( Lac) promoter is isopropyltiogalactone (isopropylt) It can be regulated by HingalaeLnside (IPTG). constructed genetics The promoter for the offspring need not come from the native osp gene. whatever Non-leaky promoters can also be used with regulatable bacterial promoters - is desirable.

This invention is not limited to a single method of genetic design. osp-i The pbd gene does not need to be synthesized in its entirety. The genetic part It may be possible to obtain it from natural sources. Identification of sudden changes in pbd DNA sequence generate accurate gene melts, as long as they are easily and accurately directed to the desired site. You may use any operating method for this purpose.

The coding part of the gene that is synthesized is designed at the protein level. , then coated with DNA. Ambiguous expressions of the genetic code are Maximize recombination possibilities to generate various distributions of amino acids in the In order to minimize the use of codons in the host cell, the engineered to allow optimal placement of limited sites to reduce It will be done.

V, F Structure consideration The design of the amino acid sequence of the barrel encoding the 1pbd-asp gene was This is related to the consideration of the above structure. The design changes somewhat depending on the GP of each type. . In bacteria, 08Ps is not an important element and therefore OSP The melting of the domain means that neither of its parental functions resides in the epidermis. There are no requirements other than that.

It is desirable that O12 does not regulate the orientation of the PBD domain. child This should not be mistaken to mean that there are no regulations within PBD. Cirl a et al. (C111R90), 5cott and Sm1th (SCOT90), and D evlin et al. (DEVL90) developed a random peptide on which phages are displayed. It is argued that the variable residue groups in the phage should not be affected by phage O8P. . Binding domains with moderate to high conformational restriction disclose a high degree of specificity They say that they will, and that a high degree of affinity is also possible. Do it like this codons for changes that specify amino acids expressed within a well-defined window. Choice is regulated. The nature of the site group is the combination of many amino acids. can be replaced over an extremely wide area. given class size The main chain conformations of many PBDs are very similar. However, in O12 Related PBD movements should not be regulated. In this way, PBD and O1 It is often appropriate to include a flexible linker between the two. like this Flexible linkers are known to have flexible regions. It can be obtained from naturally occurring proteins. For example, M13 It is thought that bacteria allow a high degree of freedom in the amino terminal domain. and has regions rich in glycine, such flexibility A functional linker may also be designed. Amino acids GLY, ANA , segments of polypeptides rich in SER and ASP have increased flexibility. I don't know if I can make it bigger. High numbers of glycines are particularly desirable.

O12 tables such as LamB, OmpA, or M13 g111 proteins If you choose to insert a PBD in the surface loop, the PBD will end at the end of O12. It does not occur when connected (a brief consideration. In such a case , O12 has some limiting effects on PBD. The end of PBD is It is placed in a somewhat fixed position. Highly altered DNA sequences on the surface Cordons that coat exposed loops and have a special binding phenotype. The osp gene can be inserted into the selected codon for the cell in question. The identified When amino acid sequences are synthesized (by whatever method), O12 constraints is lost, and the peptide has a lower affinity for the target, and and become less specific. Tan toni aiser (TANN77) , discovered a synthetic model of BPTI containing all the amino acids of BPTI, and The model is one for trypsin, which is about 107 higher than the BPTI. , has been in contact with trypsin. In this way, the modified amino The acid is a moiety of one PBD with structural constraints provided by the PBD. is the most desirable.

Amino acids adjacent to foreign epitopes inserted into LamB It is known to affect the immune properties of the body. (VAND90). Lam PBD inserted in the loop of B, OmpA, or similar 05Ps s will be influenced by the amino acids in the loop and generally by OSP. I hope that this will happen. In order to obtain proper manifestation of PBD, O12 Is it necessary to add one or more linker amino acids between PBD and PBD? I don't know. Such linkers can be obtained from natural proteins or may be designed based on knowledge of the constituent behavior of amino acids. GLY, Sequences rich in SER%ASN, ASP%ARG, and THR are appropriate. Ru. The one to five amino acids at either junction are between O12 and PBD. It will impart the desired flexibility.

The preferred site for insertion of the 1pbd gene into the phage osp gene is as follows: one of. i.e. n) where the IPBD is folded back to its original shape; b) o where the sp domain folds into its original form, and C) between the two domains. Where there is no interference.

Model of a phage displaying a single O12 amino or carboxy terminus is exposed to a solution, the exposed end of the mature OSP is i　3D model with low resolution is a good candidate for insertion of pbd gene is sufficient. .

In the absence of a three-dimensional model, the amino and carboxy termini of mature O12 are is the best candidate for insertion of the bd gene. Structural melting is the desired relationship between domains. Attachments between IPBD and OSP domains should be maintained to avoid unwanted interactions. Additional residual groups may be required. I　PBD or OS　P Random DNA or DNA encoding a specific sequence of protein homologues is If necessary, it can be inserted between the osp and 1pbd fragments.

Melting at domain boundaries within O12 is a good way to obtain functional melts There is also. Sm1th sub-clones the heterogeneous DNA into gene III of fl. During my time there, I researched such boundaries. (sMIT85).

I The criteria for identifying an appropriate O3P domain to promote the manifestation of PBD is IPB It is somewhat different from that used to identify D. Recognize one O12 Otherwise, the minimum size is not so important. Because O8P domain In does not appear in the final binding molecule and is repeated in each change round. There is no need to compose children. Concerns about raw design include: Contains. That is, a) O5P::IPBD melting promotes the appearance of rPBD. , b) the structure of the initial gene is reasonably convenient, and c) osp:: i. The pbd gene is genetically stable and can be easily manipulated. Dome There are several ways to identify ins.

Methods that rely on atomic adjustments are described by Janin and Chr+thia (JAN 185). These methods are effective between alpha and carbon (C8) distance, division of a plane (compare [103E85), or filled surface (RAS1184) matrix is used. Chn-thla and its collaborators have described the function of a large number of natural proteins with domain structures (according to their definition). We studied the correlation between R85hin is thermolysin (thermo-1y Correctly predict the stability of the domain consisting of residues 206 to 316 of (VITA84, RASH84).

Many researchers use partial tags to isolate and confirm stable domains. Protein degradation and protein sequence analysis were utilized. (For example, VITA84, (See POTE83.5COT87a, and PABO79). Pabo et al. , the cl inhibitor from coliphage lambda contains one domain that contains two domains. Calorimetry was used as an indication. They are responsible for determining the location of domain boundaries. Partial proteolysis was utilized.

The only available structural data is the amino acid sequence of the candidate O3P. In some cases, you can use arrays that predict turns and loops. Several There is a high degree of probability that loops and turns are correctly predicted. (Chou and Fasm an, (compare with CHO■74)). These sites are also i pbd genetic It is also a candidate for insertion of child fragments. .

In bacterial 03Ps, major considerations include the following. In other words, PBD is and b) that the chimeric protein is non-toxic.

From the topological model of 03Ps, the amino or carboxy terminus of O12 is exposed. You can decide whether or not. .

In that case, they are excellent choices for the melting of asp fragments into ipbd fragments. It's a choice.

The amB gene has been sequenced and is available in a variety of plasmids. (CLE M81. CHAR88a and b). I amB fragment and many other genes Melting has been used to study protein export in E. coli. various research From this, Charbit et al. (CHAR88u and b) found that the residual group of l amB was We propose a model that specifies that In other words, a) b) facing the periplasm, and C) facing the cell surface. ing. We take the model numbering for amino acids in the mature protein. I'm putting it in. According to this model, some of the loops on the outer surface are defined . They are as follows.

That is, 1) residues from 88 to 111, 2) residues from 145 to 165. groups, and 3) residual groups from 236 to 251.

Consider a miniprotein implanted in LamB. For example, G Code r N X CX RX X X CX + o S G I 2 Inserting the DNA of E,co between codons 153 and 154 of IamB It may lead to various IamBs expressed on the surface of li cells (G, , N2, S1□ and GI2 allow sufficient orientational freedom to the miniprotein. and that it can interact maximally with the target.

by utilizing isotopic enrichment of affinity (e.g. fluorescently labeled targets (through several rounds of enrichment, possibly through several rounds of enrichment), One particular LamB derivative showing high affinity for its target Insert one strand (e.g., named BEST) expressing the object. I might be able to do it. Configurations of 3 to 10 inserted residues from BEST One octapeptide with columns has a parent similar to that seen in BEST. It must have a special characteristic. The reason is that octapeptide is Lam It holds the amino acids in a conformation very similar to the miniprotein isolated from B derivatives. Because it has the internal structure that it has.

Fusing one or more new domains into a protein Generates the ability of proteins and the ability of new proteins to be exported from different cells I might. The signal peptide of the wild-type coat protein is the authentic It may work for peptides, but it is not possible to export the melt directly. be. In order to utilize the Sec-dependent pathway, it is necessary to switch to different signals. 9. May be necessary. In this way, the chimeric BPTl, 'M13 gene Vl To express and display the ll protein, a heterologous signal peptide is required. It was discovered that it was necessary to utilize phoA.

GPs displaying peptides with high affinity for the target It is extremely difficult to elute from targets, especially polyvalent targets. It might be.

(Tightly bound bacteria can easily grow in situ.) Regarding di-cleavage sites for specialized proteases (blood clotting factor ) can be introduced into the molten O8P protein so that the binding domain DNA can be cleaved from the genetic package1, and all such cleavage The phage attributed to that of has the same OSPs and thereby also even polypeptide-displaying phages can be removed from the affinity matrix without cleavage. It has the advantage that it can be eluted from the liquid. This step will be lost unless It allows the recovery of valuable genes that might otherwise have been lost. know To the extent that special the use of different proteases, or the use of phage populations with different degrees of contamination. Yes. No, and no one has suggested it.

IV, G, ii transmission synthesis This invention is limited to specific or NA synthesis or construction methods or strategies. isn't it. Conventional DNA synthesis uses altered DNA (for the production of hybrid probes). with appropriate reagent preparation for the production of It might happen.

The osp-pbd gene is expressed so that it can be expressed by an appropriately transformed GP. gDNA is present in the parent gene, such as osp-ipbd, which is It may be created by inserting. This invention is a special gDNA Method of introduction, e.g., cassette-induced or single-strand oligonucleotide It is not limited to triggers led by C.

IV, H, cloning that can be manipulated, cloning vector that can be manipulated (OCV) is the 1pbd-osp or 1pbd-osp gene of Chirella. A replicating nucleic acid that is used to introduce into a genetic package. gene patch If the cage is a virus, it may itself act as an OCV. stomach. For cells and spores, OCv can also be used for plasmids, viruses, phagemids, etc. Or maybe it's the chromosomes.

IV, I Cell transformation If the GP is a cell, the population of GPs can be transformed by converting the cell with an appropriate OCV. generated by. If the GP is a phage, the phage is a genetically engineered and infect (trams4ect) suitable host cells for amplification. G If P is a spore, then sporulation is induced, thereby OS P −P B D s will be revealed. This invention enables all cell transformations together with DNA. It is not limited to one method.

The transformed cells are initially in a non-selective state that allows expression of plasmid genes. grow under. Cells that did not transform are then selected to kill. deformed The cells are induced to express the osp-pbd gene at the appropriate level of induction. Ru. GPs using IPBD or PBDs should use the appropriate method for the GP at hand. Generally, centrifugation method to granulate (vPs) and infertility by resuspension in the medium (cells) or buffer (spores or phages) be. Whether the revealing strategy was successful or not (the entire GPs whether the affinity selection was successful or not (GPs different PB The preparations are complete for the confirmation of Ds being revealed there.

IV, J, Confirmation of revealing strategy゛ Harvested packages are coated with bacteria to determine whether IPBD is present on the surface. 1, G tested to determine which genes encode BPTI. In any (;Ps test that examines the presence of IPBD on the P surface) , ion or I. Additional factors known to be important for PBD stability Examine whether the child or AfM (IPBD) is included at an appropriate level. Tess This will be done as follows: That is, a) by labeling the affinity b) enzymatically; C) spectrophotometrically; d) by affinity separation; or e) by affinity separation. AfM (IPBD) at this stage is carried out by a chemical precipitation reaction. strong affinity for the molecule (preferably Kd<10-”M) and for wtGP. Pick something that has a small amount of affinity or no affinity.

■, - Affinity selection of target binding mutants V, A, General affinity separation techniques Affinity separation was developed for the first time in this invention to confirm whether the revealing mechanism is working. It was used. That is, the chimeric outer surface protein is expressed and the gene This test examines whether the inserted binding domain is transported to the surface of the package. is oriented such that it can be received by the target material. for this purpose When utilized, the binding domain is a known binding domain for a particular target. The target is an affinity molecule used in affinity separation steps. be. For example, one revealing mechanism is the outer surface tag of the inserted genetic package. Electrophoresis below. “Affinity substance” is a substance that has been purified and is capable of inserting NA. anhydrous, which may be confirmed by This may be confirmed by testing binding with trypsin.

When a genetic package binds to a target, the corresponding binding domain remains. You can have confirmation that it is indeed revealed by the gene package. . A package that exposes the binding domain (and binds to the target there) ) are separated from those that are not connected.

Once the revealing mechanism is confirmed, various different potential binding domains can be exposed. It is now possible to utilize an altered population of genetic packages that to determine whether binding to one or more targets is successful (or not). It becomes possible to utilize affinity separation techniques to determine. This target is , revealed binding domain, i.e. for binding with one new target one bound by a known binding domain that is a parent to one that can be selected for It doesn't have to be.

The term "affinity separation means" includes, but is not limited to: be. i.e. a) affinity column chromatography, b) affinity matrix Bulk elution from substances, C) Bulk elution from affinity substances adhered to one plate. d) Fluorescence that activated cell sorting, and e) Target substance should be present. quality, a substance that has an affinity for a substance called analyte was used to mean. In the majority of cases, the relationship between the affinity substance and the analyte is reversed. Once the impurities have been washed away, the sample is free from the affinity substance. It can be separated.

V, B, general affinity chromatography Affinity column chromatography, affinity matrix stored in some container Bulk elution from a sample material and bulk elution from a plate are very similar. Therefore, these will be treated as "affinity chromatography" from now on.

When affinity chromatography is used, 1) target substance molecules are Must be of large size and suitable solid support for affinity separation 2) the access to the matrix must be chemically reactive; After application, the target material preferably does not react with water.3 ) After application to the matrix, the target material is non-special. must not be bound to or degraded by any method, and 4) The molecules of the target substance remain unchanged, sufficient for tarp-t binding. surface (excluding atoms connected to linkers, usually at least 500A" ) large enough to connect the material to one matrix to allow There must be.

Although affinity chromatography is the preferred means of separation, FACS, electrophoresis or other methods may also be used.

The present invention is directed to cells or mice encoding proteins with desirable binding characteristics. Bacterial staff, or virus, that enriches the population for the genes that carry the virus Used for affinity isolation of bacterial viruses (or other genetic packages) It is for.

V, C, target material This invention provides a binding domain that binds to one or more target substances; or a binding domain that fails to bind to one or more target substances. can be used for selection. Of course, the peculiarity is that the binding molecule is the target substance The ability to bind tightly to a limited set of not weakly bound by other sets of target substances from the first set, or completely bound. In some cases, thermal bonding may not occur.

Target substances include polypeptides, fats, polynucleic acids, and polysaccharides. However, it is not limited to these organic polymers. However, this The invention is not limited to any of the target materials identified above. just one The limitation requires a logical coupling.

The target substance is suitable for affinity separation.

In this way, almost all molecules that are stable in aqueous solution become targets. It can be used as

Serimps such as human neutrophil z-lastase (HNE) Roteases are a particularly interesting class of potential target substances.

Serine protease is found in living organic matter [ubiquitose]. digestion, blood coagulation, fibrinolysis sis), immune response, reproduction, and peptide hormone post-translocation manipulation. It plays an important role in the process. Although the roles played by these enzymes are important, , uncontrolled or inappropriate proteolytic activity can sometimes be very destructive. It is something that has power.

V, D, immobilization or labeling of target substances by chromatography, FA For CS, or electrophoresis, the target material is combined with a second chemical component. It may be necessary to make a meaningful connection. For chromatography, the second The component for FACS is the human matrix, and the second component for FACS is the firefly. is a photopigment, and for electrophoresis, the second component is a strongly charged It is a molecule. In many cases, the target material already has the following desirable properties: Therefore, no coupling is necessary. That is, a) immobilization, b) fluorescence, or C) electric charge. In other cases, chemical or antibody-independent hydrophobic Non-covalent immobilization of sexual molecules can also be exploited to immobilize immobilized molecules used in affinity separations. or the actual target used to prepare the labeled analogue. No other substances are required. Rather, a suitably reactive analogue in the target material It might be even more convenient. Where there is no functional group to respond to The target substance can be reacted with a strong reagent solution such as a halogen. It may be possible to fix this by first creating a functional group that do not have. In some cases, the effective reactive group of the target substance may be disturbed. It may be occupying a part of the target molecule that was made to exist inadvertently. the In some cases, additional functional groups may be introduced by synthetic chemistry. .

Two general methods of immobilization are widely used. First method is to biotinylate the compound of interest. It is. The hyottinylated derivative was then transferred to immobilized avidin. Join. The second method is to generate antibodies against the target substance and use various methods to generate antibodies. immobilization and then binding the target substance to the immobilized antibody.

The use of antibodies is appropriate for larger target substances. Small target ( For example, those consisting of 10 or fewer non-hydrogen atoms) are Because the target is completely covered, a small amount of the target is exposed to the target antibody compound. be exposed to.

4.2.3. Compounds such as 3-trimethyldecane may be of benefit to alginate. is mixed with a matrix precursor such as sodium chloride, and the mixture is Even the hardening solution is extruded. The resulting beads are produced in 2.3. 3-1-rimethyldecane is sprinkled throughout and exposed to the surface.

Other methods of immobilization rely on the presence of special chemical functionality. one polypep -N　H2N　-terminal; Lysines), -C00HC -terminal: AsparticAcids; s.

-OH (Serines; Threr+n1nes; TyrOsines), to display -8H (Cysteines). Amino acid site chain reactivity For details, see CREI84. A polysaccharide has a sugar backbone. It has a free -OH group, as does the DNA that contains it.

Suitable matrices utilized as support materials include polystyrene, glass, aluminum, etc. gallows, and other chromatographic supports, and they are Produced into beads, sheets, columns, wells, and other desired forms.

Early in the selection process, a relatively high concentration of target material facilitates binding. The target concentration is later reduced to a higher Degree affinity 5BDs are selected.

V, E, elution of genetic packages supporting lower affinity PBDs. The GPs population could be interchanged with that intended for protein binding. is applied to the affinity matrix under conditions of It is divided into two parts depending on the transition of the solute gradient. parent to target The process is enriched for PBDs that are compatible with The affinity for yh is less influenced by the solute utilized. dark The condensed fraction is a variable GP that causes the eluate to be more concentrated and elute from the column. It contains s.

The eluate is a non-covalent interaction between the exposed PBDs and the immobilized target substance. 1. Preferably, the eluate destroys the genetic package. that the genetic message that corresponds to the successful mini-protein is Reproducing genetic packages rather than by in vitro methods such as PCR This is most conveniently amplified by

The list of potential eluents includes salts (Na+, NH4+, Rb+, 5O4- -1H2PO4-, citrate, K+, Li+, Cs+, H3O4-1CO3- -1Ca++, S'++, C1C1-1PO4---1HCO3-1++, Ba ++, Br-1HP04-1 and acetate), acid, heat, when combined with the target Known compounds and water-soluble target substances (or similar substances) include. Non-eluting genetic packages are targets that resist eluting conditions. DNA encoding a binding domain that has sufficiently high affinity for quality Contains. The DNA encoding such a successful binding domain can be It can be recovered in various ways. Preferably, the conjugated gene package has a change in elution status. It is easily eluted by Alternatively, culturing the genetic package in situ It might be possible.

or target with a matrix containing phenol (or other suitable solvent). Extract what is contained in the kit, and use it to generate DNA or recombinant DNA using PCR. It may be possible to amplify DNA using patterned DNA technology. Furthermore, special If a site for a given protease is engineered into the display vector, , specific proteases are utilized to cleave the binding domain from GP.

Non-specific binding to matrices and other may be identified or reduced by existing technologies.

V, F, package collection Recovery of packages exhibiting binding to the affinity column can be accomplished in several ways. You will be able to accomplish it. In other words, 1) Color with gradient as above. Collect both fractions eluted from the sample. PBD with high affinity for columns Gradient containing more concentrated GPs for genes coating s 1. 2) Collecting both components that are later eluted into the target in the form of an aqueous solution. Elute the substance through the column.

3) Immerse the matrix with the nutrient medium in water and assemble the desired package on site. Cultivate ji.

4) Remove pieces of matrix and use them to inoculate growth medium use 5) Chemically or chemically bond the target to the matrix. is enzymatically degraded, so GPs bound to the target are further eluted. or 6) degrade the package and treat it with phenol or other suitable solvent. Collect the DNA. The recovered DNA is used to transform cells that regenerate GPs. used.

A combination of the above methods can be used. Recovered from affinity matrix What we want to do is not the GPs themselves, but the information within them. survival ability Although it is strongly desired to recover the GPs, it is important to recover the genetic material. Thin The vesicle, spore, or pillion is irreversibly bound to the matrix. However, if not destroyed, cell division, sprouting, or infection may occur in the field. information can be recovered.

Proteolytic degradation of the package and DNA recovery are undesirable.

V, G, amplification of concentrated packages Viable GPs with selected binding traits can be cultivated in suitable media. or, in the case of phages, infect the host so cultured. dye. When GPs are inactivated by chromatography, asp- The OCV carrying the pbd gene is recovered from the GP and a new, life Introduced into a powerful host.

V, H, characteristics of estimated 5BDs No osp fragment for isolation of one or more clones In addition, an expression vector such that each SBD can be produced as one free protein is used. The strength of the binding may subclone the sbd gene fragment into vectors. Physical measurements can be made for each released SBD protein by appropriate methods. I might be able to do it.

If you find that the binding is still not sufficient, convert the next 5BD (new PP Determine the residual groups of BD). If the join is sufficient, a new desired join tag It is based on the expression vector that supports the genes that coat the protein.

V, l, joint selection: Molecules that bind to substance but not substance B, or bind to both substances A and B A description of the selection of molecules that bind either alternatively or simultaneously. How to do affinity separation! You can adjust it.

V, J, Manipulating Opponents It would be a shame to have an adversary for an enzyme or recipient. cormorant. Natural base or agent or molecule that prevents it from reaching the active site This is accomplished by generating. Molecules that bind directly to the active site are It may be a useful substance or a hostile substance. In this way, the following strategy was adopted. enzyme and the recipient are considered to be under the designated substance TER (target enzyme or recipient). ing.

Scientific inhibitors exist that block the active site for the majority of TERs . These chemical agents are usually useful only as research tools due to their high degree of toxicity. Ru. Two affinity matrices were prepared. That is, one has active TER, the other One utilized a blocked TER. A population of altered GP (PBD)s 5BPs that bind to both types of enzymes are selected, thereby binding to both forms of the enzyme. I obtained 5DPs that do not bind to

We expect that 5BDs will be found binding to different sites on the enzyme surface.

A pair of sbd genes are fused with one intervening peptide segment . For example, 5BD-1 and 5BD-2 have high affinity for target enzymes. binding domain, and therefore the binding is non-competitive (i.e., its Gene 6bd-1: : l 1Hker : : 5bd-2 I'm interested. It can be tested for binding and other attributes that are highly sensitive to the target. It coats the two-domains that would indicate affinity. Various 5BDs and various Generate some melts that have a unique linker. Such compounds are It has a theoretical probability that it is an antagonistic substance to the target enzyme.

Vl, development of successful binding domain compatible DNA SBD is generated using recombinant DNA technology However, the benefits originally conferred by the use of mini-proteins as one IPBD are The point is that the induced SBD will also behave like a mini-protein, and It may be possible to obtain it through scientific synthesis. (referred to as "chemical synthesis") The term, as used here, also includes the use of enzymatic agents in a cell-free environment. ing. ) The mini-protein obtained by using the method of this invention is Amino acids that do not occur naturally and those containing groups other than amino acids. It must be understood that it can be considered as a lead compound for the congeners of the series . For example, each member of a series has one substituted by its D enantiomer. A series of homologs having one amino acid can be synthesized. Also, beta Alanine, aminobutyric acid, 3-hydroxyproline, 2-aminoadipic acid, N - Congeners containing components such as ethylazurazine, norvaline, etc. can produce bodies, and they are stable, toxic, etc. cormorant.

Peptides may be chemically synthesized in solution or supporting materials. step Various combinations of fragment condensation and stepwise synthesis could be utilized.

During synthetic operations, it is desirable to protect the amino acid short chain to avoid branching. . Several different protective groups are responsible for the protection of the cysteine thiol group. It is useful for They include: That is, 1) 4-methoxyhensyl (MBzl; Mob) (NIS) 182; ZAFA88), which can be removed with HF.

2) Acetamide methyl (Acm) (NISH82: Nl5H86: 89c) in BEC, 5, mercury ion (i.e., mercury acetate) that can be removed with iodine 1 silver nitrate. and 3) S-para-methoxyhensyl (HOUG84).

Other thiol protecting groups are described in Greene's ``Protecting Groups in Organic Synthesis''. J (1981).

Once a polypeptide chain is synthesized, disulfide bonds must also be formed. do not have. Possible acidic agents include air (HOUGH84; Nl5H86), Felicia compound (NISH82; HOυG34), iodine (NISH82), Performic acid (HOUG84). Temperature, pH 1 solution, and the chemistry of chaotropy The majority of substances have multiple disulfide bonds that may affect the oxidation process. microproteins are chemically synthesized in biologically active form. So These include: That is, conotoxin Gl (13AA, 4Cys) (NISH82), Thermostable Enterotomacin ST (18AA.

6 CY s) (HOUG84), ST analogue (BHAT86), Omega -Conotoxin GV IA (27AA16Cys) (Nl5H86; R IVI87b), omega-conotoxin MVI IA (27AA, 6Cys) ((Le IV87b), 7/Iz7y--7/Toxin S I (13AA, 4C YS) (ZAFA88), muconotoxin IIIa (22AA, 6Cys) (BECK89c, CRUZ89, HATA90) . Sometimes polypeptides are naturally folded so that the correct disulfide form a bond. At other times, different removals are possible for each pair of cysteines. This should be assisted by the use of protected groups.

Successful binding domains according to this invention can bind large proteins alone or in part. For purposes including isolation or detection of the target substance, the binding protein is Used for the purpose of verifying suitability. Further aiding this purpose In order to increase the length, the novel binding protein must directly or indirectly covalently Alternatively, it may be non-covalently bound to the Rahel, to the carrier, or to the support. Not possible.

When used as a drug, the novel binding protein can be combined with a suitable carrier or may be mixed with adjuvants 1, Example I A-class microprotein design and mutagen single disulfide To obtain a library of binding domains whose conformations are constrained by Coat the DNA encoding the following microprotein genus with an appropriate OSP. Insert into the gene that is That is, X, -CX2　X3　X4　X5　CX61　1 part shows disulfide bond are doing. Disulfides are usually between consecutive cysteines in a polypeptide chain. Not formed. X above. One or more of the residues marked with are novel. It has been greatly altered to obtain the bond. Prefix XI or X6 There is one or more amino acids that follow, however, before or after The residual group after X6 is not significantly constrained by the disulfide bridge represented in the diagram. and it is not advantageous to replace these remote unbridged residue groups.

The last X residue is linked to osP of the genetic package.

Xl, X2, X7, X4, X9, and X can be transformed independently, i.e. , one different scheme of variation can be utilized at each site. XL X6 is a minimally constrained residual group and changes less than other sites.

For example, Xl and Xh can be one of the amino acids (E, ni, T, and A) This cent amino acid is desirable for the following reasons. sand i.e. a) positively charged, negatively charged and neutral amino acid possibilities are provided; Ru, b) These amino acids are codon RMG (R = equimolar A and GSM = equimolar A and C) and C) These amino acids are accepted for appropriate processing by a signal peptidase. Ru.

In one preferred example, X2, Xl, X4 and X5 are part of the alternative set (F , S, Y, C, L, P, H, R% I%T, N.

V, A, D, and G) respectively coded by the cordon NNT. It is only by being changed that something changes.

The advantage of the NNT cordon over the NNK cordon is that the replaced cordon As the number of dons increases, it gradually becomes clearer (because the five chords library whose code was replaced by either an NNT or NNK cordon. Table IO and Table 130 are compared. NNT combines 15 different amino acids. It encodes only 16 DNA sequences.

In this way, the amino acid sequence of 1.139.10', no stop, and There is a DNA sequence of 1,678.10'.

A library of 10' independent transformants contains 99% of all possible sequences. I'm reading. The NNK library contains 6,4,107 sequences, but the complete Sampling requires a larger number of independent transformers.

This sequence utilizes the natural M13 gene III promoter and signal sequence. It is manifested as a melting of M13 with gene III protein. Residues 16 to 2 The sequences of M13 gene III proteins up to 3 are S, 6HS, AE, T VE is 21, and the signal friend Petit d'Ace I splits after S16. child The segment is replaced with: That is, SL 6 G A ls A E GX + CX 2 X v X 4 X , CX 6S Y IE G RV ITVE0 The replacement of H, S, with GA restrains the phage for infection. Please note that there is no. One difference between PBD and mature protein III. It is possible to insert two bobbin F, Xa recognition/cleavage sites (YIEGR/Vl). It is for use.

This not only allows positioning freedom relative to the PBD, but also It also allows for dehiscence.

Xl, X2, X5, and X6 are NNT (F, S, Y, ClLSPSH% R, VSTSN, V, A, D, and G) one phage library, and X3 and X4 are NNG (L, S% W, P, Q, R.

M, T, and V, A, E, and G are allowed) One phage library is named TN2. Lee is approximately coated by an approximately 1.5 x 10' DNA sequence. Reveals 8.55X10' microproteins. , NNG is the third and third To exclude the possibility of at least partially cysteine at the fourth site, the third and fourth is used at the variable site (the central site of the disulfide-confined loop).

Devlin et al screened 107 transformants. Each of them is streptavidin 1012 random bentadecapebites can be revealed for affinity with.

And they have 2o strings with eight unique sequences (rAJ - rlJ). A leptavidin-binding phage isolation material was discovered. All isolated substances are available on HP; 15 /20, HPQ; and 6/20, included HPQF, but they were They were located in different parts of the body. The most frequently encountered isolated substances were They were D (5), I (4), and A (3), which were completely missing tin. deer However, the two positive isolated substances, rEJ (1) and rFJ (2), are a pair The formation of disulfide bonds was possible because it contained a cysteine in the there were. The sequences of these isolated materials are shown in Table 820.

TN2 library has the potential to express streptavidin binding activity. One estimate that is very similar to Devlin's rEJ and rFJ I am aware that I should have included the microprotein HPQ. , HPQ consists of the AEG amino acid terminal sequence common to all members of the TN2 library. The sequence P has the potential to form disulfide bridges in four spans. Followed by CHPQFCQ, one serine (S) and one bobbin factor Xa Followed by recognition sites. (YIEGR/IV) (See table 820) . The high-lot experiment involved the association of streptavidin with the HPQ-carrying phage. The composition is comparable to that of Devilin's rFJ isolate, and both The margin of was above the background (1,7X). Therefore, fixed The TN2 library against streptavidin was screened.

Streptavidin has a specific activity of 14.6 units (1 unit) per lag. A free protein (Pier ce). in PBS containing 0.01% acidide. 10g of stock solution is made per milliliter. 5trAvs Totsuku's 100mm, -L is 250 of 1m-mulon (#4) plate The cells are added to a volume of 1.5 L wells and incubated overnight at 4 degrees Celsius. stock removed and 250 mu of PBS containing BSA at a concentration of 1 mg/a+L. L and kept at 4 degrees Celsius for an additional hour. Phage binding assay Wells should be treated with 0.1% tween before use in Wash quickly 5 times with 250 μL of PBS containing water.

One known ff1 (Tn2 library) was added to each 5LrAv-coated well. Add 100 μL of binding buffer containing 10” pfu’s of phage. Ru. Continue culturing at room temperature for 1 hour, then remove unbound phage and incubate with PBS 0. Wash quickly 10 times with 1% Tween. Furthermore, the pH is 7. Wash with citrad in steps 6 and 5 to remove non-specific binding.

Bound phages were mixed with 1 g per mL BSA and 60 ml of 1M Tris pH 8. - with 250 μL of pH 2 Citrade buffer containing a neutralizing agent with Elute. The eluate is used for subsequent rounds of binding, washing, and elution. used to infect bacterial cells to generate new phage stocks. It was. The capacity building cycle is repeated twice more (three times in total) and then numerous times. Individual phages were added to the array as clonal isolates and also tested. . The number of phage present in each step is appropriately diluted and contains E. coli. The black forming unit (pf u's ) is determined as

Table 838 shows the peptide sequences discovered to bind to St rAv and the last (Round 3) Indicates the frequency of random picks taken from the phage pool There is.

Intercystine segments of all putative microproteins examined were HPQ It contained a motif. The variable residues before the first cysteine are (F, S, Y, C ,L,P,H,RSI.

The residue groups selected were +Y, H, D, Nl, and phage HPQ was P Variable residue groups after the 1st distinence having (F,S,Y%C,L, P, H, R, I, T, N, V.

I was able to have A, D, and Gl. The selected residual group is +P.

S, G, R%V), and phage HPQ has Q. relatively The missing binding phage HPQ is probably due to P4 or QIj or both. I don't know.

In a control experiment, the TN2 library was screened in the same manner as shown above. The target protein is the blockade agent BSA. has quality. After three rounds of binding, melting, and amplification, 16 lander Muphage Black was selected and sequenced, and half of the clones had insertions (8/8). 16) was not seen. And for the rest, the array shown in Table 839 was there. There is no consensus in this collection.

A phage-related microprotein, HPQ6, was revealed. CHPQ It is identical to HPQ except for the replacement of FC with CHPQFPRC. (table 820).

When manifested, HPQ6 is associated with either HPQ or Devlin rFJ isolates. had a substantially stronger affinity for streptavidin than . (The rEJ isolate of Devilin was not studied.) Notiothreitl ( (dithio-threitol) (D among the revealed peptides. suggests that one disulfide bridge present in is necessary for good binding. ing. Examining the results of the TN2 library screening, phage HP The bond of Q6 can be obtained by replacing P4 with one of (Y, HlL, D, Nl). Or Q+, + (2%S1G.

The tree can be further improved by replacing it with one of R and Vl.

Example II A CH3::HELIX::TURN::5TRAND::CYS UNIT The parent class 2 microprotein is probably the naturally occurring class 2 microprotein. It is of good quality. It also meets the criteria for Class 2 micro-proteins. One large tank whose structure may be satisfied or partially modified. It may be a fake domain. Some changes are made using one cysteine (or It could be something as simple as introducing a pair of cysteines into the base of a hairpin structure. unknown, and by doing so the hairpin is closed with disulfide bonds. It will be. Alternatively, some intermediate residue groups may be modified to achieve a hairpin structure. Further, it will likely be closed in a more elaborate manner. Its parent class 2 Mi Proteins are also two or more naturally occurring proteins. i.e. wax to alpha of one protein and hetas of the second protein It will be a hybrid of structures from Trant.

One potential use is for microprotein motifs to be used in each Disulfide loop structure surrounding the base, turn, and return strands It is to accomplish. Such structures can be designed or constructed using known tertiary It would be possible to obtain it from the protein in its original structure. Scorpion neurotoxin, deformed Substance 3, (^LMA83a, ALMA83b) (hereinafter referred to as 5corpTx) ) contains the structure shown in Figure 1, and it contains one wax (from residues N22 to N33), one turn (from residues 33 to 35), It consists of one return (residue groups 36 to 41). 5corp TX connects residues 12-65, 16-41, 25-46, and 29-48 Contains disulfide. CYS25 and CYS41 are very close together. can be linked by disulfides without disturbing the main chain. Figure 1 shows CYS25 and cys4. It shows the combination with. Furthermore, c y s 2. is converted to GLN. is being given.

One disulfide forms between 25 and 41 and Ricks expects to form. The amino acid sequence shown corresponds to this structure. Has a high degree of compatibility.

The presence of GLY, 5, GLY 36, and GLY, To accommodate the changes required around CYS41 to form a turn, This gives sufficient flexibility to the stretched strands.

From the inspection of the structure (Bronkhaven Protein Data Bu (as found in 1sN3 of nk), the set of residues below are for change I think that would be desirable. That is, set l 'A group codon permissible amino acid Naa/Ndnal) T27NNG LzR’MVsPTAQKEfG 13/152) E2. VHG LM VPTAGKE 9/93) person,, v) l to LliVPTAGKE 9 /94) [32VHc LMVPTAGKE 9/95) G24 NN G LIR”MVSI’TAQKEWG13/156) E23 VHG LMVPTAGKE 9/97) Q34 VAS HQNKED 6/6 Note: Amino acid index indicates the diversity of codons.

Positions 27, 28, 31, 32, 24, and 23 are.

It forms one side of the helix. In each of these areas, the following changes occur. I picked up the cordon. That is, a) those containing the parent amino acids; b) The set of residues that have a predominance of residues that make helices desirable. C) provides a wide variety of amino acids, and d) Something that leads to a distribution that is as average as possible. Position 34 is the turn part 9 , 9 - Site group of 34 residual groups 27.28.31.32.24 and can interact with molecules that contact 23 site groups. In this way, 1° which tolerates this change and provides amino acids compatible with the turn. The changes were 6,65,106 coated by the 8,85,106 DNA sequence. 3 amino acid sequence.

set 2 91 groups Cordon Allowable amino acids Naa/'Ndna1) D211 1/ HG Ll1MVIP2T2A'HQNKDE 13/182)T? tNN G　L”R2MVSPTAQKEWG　13/153　)　k:+o　)If; KEQPTALMV 9/'94), +,, vuc; KEQPTALMV 9. -'95) N4. VHG LMVr’TAGKE 9. -'96 ) sly RR’r 5NDG 4. /’47) Yis NHT YSF HPLNTIDAV 9. /9 Bojon 26.27.30.31 and 32 are substituted to increase the capacity of wax-friendly amino acids in the population.

Residue groups 37 and 38 are in the return strand, so other change cordon Adopt. This change is 4.43.1063 amino acid sequence and 7.08.106D One library embodying this scheme thus allows for NA sequences. Lee can take samples very effectively.

Example 111 Class 3 microprotein design and mutagens Two disulfide bonds A microprotein with a disulfide bond between two parent microproteins that have a bond The substances are alpha-conotoxins, namely Gl, G■A%Gll, Ml, and It may be possible to use Sl as a model.1. These are saved below. 2, which has a similar structure, Hashimoto et al. (HASH85) Kishin G1. report on the synthesis of 24 analogs of GlI and MI.

. Numbering strip for Gl (position 2.3.7, and CYS in 13) Using schemes 4, 8, and 8, which allow proteins to become toxic, Hashimoto et al. 10 and 12 are reported. Almquist et al. (ALMQ8' J) is (d e s G L LI +) alpha-conotoxin Gl and 20 We synthesized an analogue of . They replaced GLY in place of PROs and created two different and raised a sexual body, possibly related to a different disulfide bond. reported. They allowed some alternative substances that allowed the protein to be toxic. was found between residues 8 and 11.

Zararalla et al.8ZAFA88) found that the PRO replacement in position 9 discovered that it provides active protein. Each group cited is An in vivo toxicity test was utilized as an assay for chemical reactions. Is that kind of research? Although it can be inferred that the active protein has the three-dimensional structure of the parent, It cannot be inferred that the three-dimensional structure of the inactive protein parent is lacking.

Pardi et al. (PARD89) reported that alpha-conotoxin Gl was determined by NMR. It was determined that it could be obtained from Venom. Kobayashi et al. (ni0BA89) ”) reported the three-dimensional structure of synthetic alphaconotoxin G1 from MMR. The data match the PARD89 data. Citing Figure 5 of Pardi et al. ing.

The residual group GLU is a known analogue or congener of GLTJ, ARG, mole It is known for facilitating LE. The desired change codon is the amino acid set. This is an NNG that allows stop (L'R2MVSPTAQKEWG<stop>).

From Pardi et al.'s 5, the site group of GLU is from residue group 9 to 12. It can be seen that it is projected onto the same area as a strand consisting of . residual group 2 and 3 are cysteine and cannot be changed. The side group of residue 4 is , from residue groups 9 to 12 (point awayX distracts).

In this way, this residue transformation is deferred until subsequent rounds. PRO5 is It may be necessary to cause the proper disulfide to be formed. G If LY is substituted, the peptide is now woven into two forms, but There is no toxicity either. PROl is allowed to convert, but in the first round I can't even think about it.

No substitutes for ALA6 have been reported. One desirable cordon is AL A, THR%LYS, LrGLU (small hydrophobic substance, small hydrophilic substance, plastic This is an RMG that produces a CY　S　7 is not converted. A Homologue proteins with LA are toxic, but GLY remains intact. Homologous proteins with various amino acids at position 9 Quality is toxic. In this way, Fs2YCLPHRI TNVZDG is allowed. The NNT variation cordon of -n is used. In position 14 after the 40th YS , A.L.A.

THR, LYS, or GLU (via RMG cordon) is allowed. this The change is 1,053.107 encoded by the 1.68.10' DNA sequence. Accepts amino acid sequences. 2.0・107.3.0・107 and 5.0・1 A library with 07 independent transformants each has approximately It reveals 70%, almost 83%, and almost 95%. Dew. Other variations are also suitable. Regarding alpha conotoxins, especially AL11Q89, CRLIZ85, GRAY83, GRAY84, and PAR See D89.

The parent microprotein was designated by Pease et al. (PEAS90) “Hybrid I” and “Hybrid Ill” can be substituted for one of the proteins. Maybe. Compare with Figure 4 of PRAS9G. For any protein One preferred set of highly variable residue groups consists of: sand In other words, it has changed and is acceptable. Parent amino acid Codon Amino acid AA / DNA sequence A5 RVT M) GT NS 6 / 6P6 VYT PTALrV 6/6 E7, RR5EDNKSRG! 7/8T8 ■G TPALMVQKE 9/9A9 VHG ATPLMVQKE 9/9Am ORMG AEKT 4/4 K12 VHG KQETPALMV 9/9Q16 NNG L’R”S, W PQ Kuchini G 13/15 This is coated by the 1.26.107 DNA sequence. The resulting 9°55.106 amino acid sequence is provided. Consists of 5.0.107 conversion body One library allows expression of 98.2/<- cents of all possible sequences. do. At each position, the parent amino acid is acceptable.

Position 5 provides an amino acid compatible with one turn. position 6, ILE and VAL have branched beta carbons, and the chains are ridged. ILE and VAL are allowed for this reason. At position 7, helix ami Allows ASP, ASNo and SER, which are often expressed at noic acid termini. Posisi In versions 8 and 9, the charge is different because it is an inherent part of the helix. and some heliophiles that have hydrophobic organisms. X-friendly amino acids (ALA%LEU, MET, GLN, GLU, Soshi1:LY S) is allowed. Position 10 is at the far end of the helix, so Allows type sets ('ALA, THR, LYS, and GLU).

This set includes three helix-friendly amino acids as well as extremely well-tolerated amino acids. However, it contains THR, which allows positive, negative, and neutral affinities. 1 Side groups 2 and 16 are cast into the same region as the residues already described. shoot

At these sites, there is a skew toward helix-friendly amino acids. It tolerates a wide variety of amino acids.

The parent microprotein has residues 9-24 and 31-40 of aprotinin ( aprotinin) and two disulfides (Cys9-C One polypeptide with ys22 and Cys14-Cys38) It may also be replaced with de.

Such polypeptides have the same disulfide content, such as alpha-conotoxins. and the two bridges have spans of 12 and 17, respectively. It is something that exists.

Residues 23, 24, and 31 are from the amino acid residue set (G).

S, R, D, N, H, R, T, A), so the necessary A sequence is discovered that encompasses a geometric turn. BPTI in the P1 region reveals microproteins folded into similar stable structures Use trypsin or anhydrous trypsin to enrich GPs with affinity molecules. use

The three disulfide-bonded parent microprotein corn snail (Cnnus) , 10-30 amino acids in length, and particularly rich in disulfide bonds They therefore produce conotoxin (C1111(Y+xins)), It is a microscopic protein. A novel microorganism with three disulfide bonds The protein is also mu (GIIIA%GI IIB, GI Irc) L<ill ga-(GVIA, GVIB, GVIC, GVllA, GVIIB, M VIIA, MVllG and other nchonotoxins could be modeled. ,, muconotoxins have the following conserved structure, i.e., muconotoxins have the following conserved structure: - The non-three-dimensional structure of conotoxin has already been disclosed.

Hidaka et al. (HIDA90) established a disulfide association. Diagram below Lamb is gengraph-toxin I (mu - also known as conotoxin GIIIA).

The connection from R19 to C20 can go to the strand from Q14 to C15. can. One form of desirable change is to change the residue group in one loop. 1. The longest loop contains only five amino acids, so it forms a loop. It is also appropriate to change the residue group connected to the cysteine. For example, Residues 5 to 9 can be replaced by adding 2.11.19 and 22. May. Another useful change is to change the residues 11-14 and 16-19 to , it would be possible to change eight amino acids. Regarding muconotoxins For BEC, 89b, BECK89c, CRUZ89, and old DA90 checking ...

Omega-conotoxins would be labeled as below. King kelp pepti has the same disulfide assortment as omega-conotoxin, but has a different It has biological activity. 1londvard et al. (I100D90) , C, and tcxtilc report the sequences of three homologous proteins. mature Only cysteine is conserved within the toxin domain. The cysteine spacing is Although completely conserved, other bones have identical amino acids in all three sequences. not, and only a few positions show something like a pair.

In this way, all positions (except cysteine) have one stable distle. will be freely substituted with a high probability of forming a fido structure. I conclude that Regarding omega-conotoxins, HILL89 and 5UNX8 See 7.

Other microproteins that may serve as parental binding domains are Eggplant maxima (Cucurbit maxima) trypsin inhibitor 1 ( CMTr-1). CMTI-111 is also suitable. These are summer squash, Capocha serine protein containing inhibitors from zucchini and cucumber Men/<- of the tease inhibitor. (VIEC85). McWherter (M(J)+89) are human 0 leukocyte elastane Kapocha species protease inhibitor with affinity for Cathepsin G and Cathepsin G. Describes a deformable material with a synthetic arrangement of materials. Of course, any member of this genus Members could also be used.

CMT I-1 is held in a fixed conformation by three disulfide bonds. The smallest protein known to contain only 29 amino acids It is one of the proteins. Its structure +, t, X-ray diffraction (B(lDE89) tN Using both MR (IIOLA898 and b) (7), Bode and its cooperation are being studied by people. , CMTl- [ has a bale-like shape, and it has a helical shape. It does not have a cus or heta sheet, but it consists of a turn and and are connected by a short polypeptide extension. Disulfide bearings are C ys3-Cys20, CyslO-Cys22, and Cys16-Cys28 It is. CMTI-1: tryps studied by Bode et al. In the in compound, 13 of the 29 inhibitor residue groups are directly linked to trypsin. have a relationship.

The majority of them contain the chemical reaction site binding Arg5(PI-)IIe6 In the important connecting segment Va　2(P4)-G　u9(P4') one that exists and was also found in other surin proteinase inhibitors. Conformation - Exists in a constellation.

CMT I-1 is a single tube for trypsin of approximately 1.5.10-12M. I have 1. McWherter et al. suggested an alternative substance for "moderately abundant hydrophobic groups" in They are Residual groups that provide detectable binding to HLE (HAL, ILE, LEU, A found an extensive set of LA, PHE, MET, and GLY). cathepsin As for G, they are truly desirable abundant site groups (particularly the aromatic genus K). I was expecting that. They believe that PHE, LEU, MET, and ALA are their bases. found to be functional according to standards. They are TRP, TLRl or H I haven't tested IS. (ALA uses the second smaller available side group Note that the first desired change strategy is to remain Group ARG, , VAL2, PRO4, ARG, , ILE1LEU7, METs, G LUe, L Y S + +, HIS25, GLY2 TLY, and GLY 2. It means replacing part or all of. For example, if the target is HNE In some cases, it is possible to synthesize DNA that has the following possibilities: sand Well, Parent Vg codon Permissible amino acids #AA/#DNA5eqsARG, VN T R3LPHITNVADG 12/12VAL2NIIT VILFYHN D 8/8PRO4VYT PLTIAV 6/6ARG5VNT R8LO HITNVACG 12/12ILE, NNK 20 all 20/311E LI, VWG LQMKVE 6/6TYI? 2□ NAS YHQNKD E? /8 This was encoded by a DNA sequence of approximately 1.03.10' Approximately 5.81.106 amino acids are allowed. Is it an independent converter of 5.0/107? A single library of Ru. Other variation schemes may also be utilized.

Other inhibitors in this category include: That is, C1trullus Trypsin inhibitor 1 (OTLE87) from Vulgaris, Bryo Trypsin inhibitor 11 (OTLE87) from Nia dinica, Cu Trypsin inhibitor 1 from curbita a + axima (OTLE87 ), trypsin inhibitor 111 from Cucurbita maxima ( of OTLE87), trypsin inhibition from Cucurbita mnxi+na Substance IV (OTLE87), trypsin inhibitor from Cucurbita pepo Harmful substance +1 (OTLE87), trypsy from Cucurbita pepo Inhibitor l11 (of OTLE87), from Cucumis 5ativus Trypsin inhibitor 11b (OTLE87), Cucumis 5ativu Trypsin inhibitor TV (OTLE87) from s, Ecballiumel trypsin inhibitor 11 (FAvE89) from aterrium and Inhibitor Cto1 (OTLE87) from Momordica repens Other microproteins that can be used as phase potential binding domains include Kuraka's intestinal toxin inducers E, col i, C1trobacter fr eundii, and other bacteria-induced thermostable intestinal It is a toxin. These microproteins are secreted from E. coli. is known and has sufficient stability. Synthesis of these proteins, cloning See below for information on structuring, expression, and characteristics. That is, BH AT86.5EK185.381M87, TAKA85, THOM85a to b, YO3H85, DALL90゜D11AR89, GAR187, GUZlj8 9, GUZM90, H01JG84, UBO89, KUPE90° Oka87, OKAM88 and OKAM90゜Example IV CU(II+, 1, 2 histidines, and - 1 methylsni) A mini-protein with one cross-link consisting of The sequence as below is i.e. HI S-ASN-GLY-MET-Xaa- Xaa-Xaa-Xaa-Xaa-XIILI-HI 5-ASN-GLY-C YS and CYS-ASN- to LY-MET-X88-Xaa-Xaa-Xaa -Xaa-Xaa-HIS-ASN-GLY-Hls is not shown in Taiagelum. In order to form a certain structure, the bond with Cu(If) is made into a container 1゜ Other combinations of Hls, MET, HIS, and CYS along the chain are also similar. It appears to form a similar structure. Positions 2 and 3 and positions 12 and 13 Certain amino acids ASN-GLY are confers an amino acid carrying a metal-binding ligand with sufficient flexibility for this purpose. other may also be used, i.e. GLY-ASNlSER-G LY, GLY-PROlGLY-P RO-G LY, or PRO-G LY-ASN may be used 1. Also, the first and second or third and fourth replacing one or more residual groups in the loops connecting the metal-binding ligands of Both are possible. for example, must form the structures diagrammed for the various amino acids at Xaa4 . It is because the side groups Xaa4 and Xaa6 are close together and the miniprotein can be expected to exist on the surface of

Since the changed amino acids are retained, their flexibility is limited. child cross-links are somewhat different from disulfide linkages. C Al The separation between F, and Calpha,1 is similar to the separation of CalphaS of one cysteine. It's a big deal. Furthermore, residual groups 1 to 4 and 11 with metal ions The interaction between residue groups 4 and 14 is caused by the disulfides of residue groups 4 to 11. It is expected that movements from 10 to 10 will be further regulated. single thistle Fido bonds exert a strong remote constraint on the alpha carbon of the attached residue; For example, the vector from N to C in the main chain has a very small amount of directional restraint. Or don't let me work.

For the desired sequence, site groups from residues 5 to 10 are targeted form a special interaction with Other variable amino acids, such as 4.5.7, if 3 is appropriate.

Large spans may be utilized in the following cases. i.e. encompassed The alpha helix whose sequence regulates the conformational freedom of the polypeptide backbone or other secondary meanings are cells that have a high potential for forming structures. This is the case when it contains a component. Mini protein with four CYSs The quality can form three special bearings, two HISs, one A miniprotein with MET and one CYS has only two Only two special compounds can be formed. These two structures are mirrors through Cu. They are related by plane symmetry. Since the two Hlss are distinguishable, the structure The structure is different.

If such a metal-containing miniprotein is displayed on a filamentous phage, the phage Cells producing phages can also grow in the presence of appropriate metal ions. Alternatively, phages may be exposed to metal only after they have been separated from cells.

A mi with one cross link consisting of ZN (11) and four cysteines. Ni protein Example V Cross-links similar to those shown in Example XV include zinc finger proteins. quality (G lB588, GAUS87, PARR88, FRAN87, Cl0f87 , Soshite HARD90)i:, is thus exemplified. A genus of zinc fingers , Zn,, (PARR88, FRAN87, Cl0f87, EVAN88, BERG88, CHAV88). I have a YS and two HIS residuals. Gibson et al. (GIB588) We studied several sequences thought to form zinc fingers and found that these A three-dimensional model for compounds is proposed. Most of these arrays are retained There are two CYS and two HIS remaining in the positions that were held, but some It has three CYS and one HIS residual. Gauss et al. (GAUS87 ) also has three CYS and one HIS residue binding zinc. reported lead finger proteins. Hard et al. (HARD! 110) One tab consisting of two zinc fingers each having four CYS residues Reports the three-dimensional structure of proteins. All of these zinc-binding proteins are Stable in a reducing intracellular environment. One CYS::Zinc crosslink One desirable example of a mini-protein is the arrangement shown in Figure 1 of HARD90. It consists of the remaining groups 440 to 461 of the column. Residues 444 to 456 were changed Profitable S fish 444S plate, kite 2/2 AsP445 Super P, Li1N, GLU, LYS 4/4GLU44 g GLU, LYi9. G Go 3/3ALA447 MiA Pa..., GLY , 91! λ 4/4 Sli14411 1igR, ALA 2 / 2GLY 449 GLY, Sn, ASN, ASP 4 / 4CYS450 CY S, Pn, ARG, LEtl 4/4FIXS451HXS, GX M, A! 9N, LYS, AjP, GLU g/6TYR452'n 'R, PIE, [S, L decoy 4 / 4GLY453 GLY, 81! R, Super N, ASP 4/4 VAL+454, VAL, ALA, ASP, G1 ．． +Y, SRR, ASN, TER, Engineering LE’LXrJ455’Lam1. Revised S, ASP Hau4/4 This encodes the same number of amino acid sequences 3, leading to the 77/107 DNA sequence. 1.0.1 with 108 independent transformers One library would reveal 93% of the accepted sequences. 2.0 A 10' independent transformer will reveal 99.5% of the allowed alignments .

Table 2 Desirable outer surface proteins Gene Desirable outer surface Reason for priority puff cage protein M13 coat protein a) exposed amino terminus, (gpHIII) b) pre-coat protein Possible post-conversion steps C) multiple copies in pillion d) Melting data available gplrr a) Melting data available b) Amine-terminated exposed C) Motion examples available Ph1XIT4 G protein a) b known to exist outside the pilion ) is small enough that the G-jpbd gene can replace the H gene E. coli LamB a) Melting data available b) Not important 0rnpCa) phase model b) Not important, there are many OmpA a) Phase model b) Not important, there are many C) Homologues in other general matters OmpF a) Phase model b) Not important, there are many 0rnpE a) Phase model b) Not important, there are many C) Inducible B, 5ubtilis Cot Ca) Spore manipulation after conversion b) Tapping on spore coat Special arrangement where proteins are localized C) not important CotD Same as CotC Table 10 Presence A obtained from Mr. Qi VG cordon, MJ converted fxS near -ton, CD) + (E) = (K) 2 (R) [D] + [E] Cabinet [Kl + [ R] -,12ratio -Abun(W)/Abun(Sl -0,407 4 Table 10 Abundance obtained from various g-cordons (continued) [D] + [E] -0,L162 [Kl + [R] -0,1264r & io -Abu n(Kl　/Abun(5i)　-0,41176Table 10 Various g cordons Abundance obtained from (continued) C1J converted NNT g 64.0. 015625 1゜ 7 1213.0. 0078125　1゜Table 10 Input from various Vg cordons Obtained abundance (continued) D, optimized NNG Table 10 Existence layers obtained from various g-codons (continued) E-N not optimized NS (NHK provides the same distribution) B Table 130 Sampling A of the library needed by fNNK)'', each Class edge peb 4-1' number eocal g 64,000,000 5eo p-free 5equ@ncas.

Sampling of libraries encoded by Table 130 (NNK)’ (continued) B. Any stopless DN A array from one defined class to Probability of encoding a xapeptide Sampling of libraries encoded by Table 130 (NNK)’ (continued) C1 Different stopless applications in each class with different sizes expected. Number of amino acid sequences Libra75ize m1. oooOE+06eoeil -9,7446 E+05 k gajrIpled -1,52Librir7! liZ@! m 3.0OOOE+06total -2,7ali15 +06, * I! 5unpled -4,36 Table 130 (NNK)' Coded by Library sampling (continued) Libra:yBIZe-1,oooOE+ 07toeal -8,1204E+06 wisdom sampled -12,69L ibra75ize s-3,0OOOE+07hiojai-1,8633E +07 k sampled -29,11 Copied by Table 130 (NNK)' Sampling of the downloaded library (continued) Lik)ra-/5lice-7 ,6000E+07Libra:ysize w 1. oooOE+08shioe aL # 3.6537E+07’t sampled m 57.09 Table 1 30 (NHK)’ Sampling of the library coded by (continued) Li b: a=y 5ize -3,0000E+08 ozHyu -5,2634E +07k sampled -82,24Lib=) 了5ize wa'J -,0OOOE*09coral -6,1999E+07% sample d-96,878130 (NNK)' of the library coded by Sample collection (continued) I, 1b=a+: Ysize Iw 3.0OOOE+09j ana1-6.3B90E+07% sampled -9S, 83 Table 13 Sampling the library coded by 0 (NNK)’ (continued) by D Formula for the second counted quantity in the table Table 131 Library samples encoded by (NNT)” (NNG)’ collection X is FSS, Y, CSL, PSH, R, r, T, N, V.

A, D, and G are also fine.

"L2, R", S, W, PSQ, M, T, V, A, E.

G is also fine.

The library contains the 8.55.106 amino acid sequence and the 1.47.107D N A sequence. Consists of columns.

The total number of possible aa sequences is 8,555.625.

x LVPTARGFYCHIND S θ　VPTAGWQMKES Ω LR The first, second, fifth, and sixth positions hold X or S. Third part The fourth position holds θ or Ω. The array is xs, Ss, θS and so on. and are summarized in ΩS.

The following table shows that any special NA array falls into one of the defined classes.

Lib:ary 5ize -1, OSampling -, 0OOO1tTable 1 31 (NNT)’ (NNG)” Tori (continued) The next part is how many arrays in each class can be expected in a library of different sizes. It shows how.

Librarysize-1, oooOE+05Library 5ize Q 1. oooOE+06Librarysize -3,0OOOE+06Table 1 31 (NNT)’ (NNG)” Tori (continued) Libr, ary 5ize -11,5556E+06Library 5i ze -1,oooOE+07Library 5ize m 3.0OOOE +07 Table 131 Library coded by (NNT)’ (NNG)” Sample collection (continued) SSOΩ5S,,,,,4.0(100,01Libra:ygizem1 ．． 0OOOE+08 Table 132 Relative effectiveness of various simple change codons Number er of codons ζ 7 IDNλ/:AA #DNA/:AA:jDNA/1tAA[#DNA][ i:DNA] [#DNA]va “” NNK 8.95 13.86 21.49assuLrrg (2,86・1 0 勺 [84740 towns [2,75・10”] 5eops vanish (3 ,240'l (6,440') (1,2E140') NNT 1.38 1 ．． 47 1.57, 03.106] [', 6B.10] [2,68.10 8] (7, 55・10') (1, 14・10') +L7L・10s) NNG 2.04 2.36 2.72assuming [7,5940'] [1 ,1440'] [171・10"1seops vanish (3,7-x O’l (4,8340’) (6,27・lO’) Table 155 Octapeptide The input between the alpha cordons of ffiMEx ended 5tran d: angle of C11-(, 2-C, 3-138' Reverse eurn beews@n residues 4 and 5゜Alpha helix: angle of C, 1-C112-C, 3w 93'3 5 ．． 5 3.8 + 4 5.1 5.4 3.8 + 5 6.6 5.3 5.5 3.8 -6 9.3 7.0 5.6 5.5 3.8 -7 10.4 9.3 6. '5.4 5.5 3.8 -8 11.3 10.7 9.5 6J 5.6 5.6 3.8 - Table 156 type Al in the closed miniprotein of disulfide cyclo (CXXXXC) Distance between carbons: 1nCI! 4 S, G 6.0 3.8 + 6 4.8 5.3 5.1 5.2 3.8 -3 6.3 34 - 4 7.5 g, 4 3.8 - 6 5.6 7.5 7.7 6.4 3J -36,73,8- Table 820 Peptide phage 7m 1 bioeic Dev(El　λE　-LCHPQFPRCNLFRKVPPPPPPAE,, .

)IPQ6　λEGPC)iPQFPRCYIEGR/:V------E,, .

Table 838 Streptavidin-linked disulfide-restricted peptide #2 glu g lytyr ays his pro gin pheeye pro ser 4#4 glu gly his cyg his pro gln phe eys ger ger 3#5 glu gly l@u ays his pro gin phe Cys gly ser 2#8 glu gly asp cys his pro gin phe ays ser ger 2#1 glu qIY&Sn C’1shis pro gin phe cyl pro! iar#3 glu gly asp cys his progin phi ays arg sar 1#L] glu (il ”/&Sp cyg his pro gin phe eye val g er　ICys　his　pro　gin　phe　cym　consensu s table 839 Array 1 manually created by BDA11 reduction a = ayV vvv v　V　9-? a6V#21 glu gIYgly eye phe ly s arg asn eyg CYr Bel: L#22 glu gly his ays asp 1y111ye ile ays leu ger l#23 glu gly phe cym his ehr ala ala cys phe ser M#24 glu gly his ays ty r lys glyval eye sar sar 1#25 glu gl y　his　ays　asp　lys　serp　arg　CYJI　pros er　＃26　gLu　gly　iie　cys　tyr　arg　leu　 asp cym ile ser l #27 glu gly gly ays phe pro trp his ays ph= ser 1828 gl u　91y! jar C7m! Lap tier l@u arg C7tl ! ! p tier LNo C0n5enSu! 9　observed.

literature AL! 1iA 83a: Almassy, child C, JCFoneec±lla-Camps, FL Su ddamTh, ELndCE Bugg.

σMo1BLC=(19831,PutU,:4972-[-Baker, K, N Mackrnan, and 3 Halland.

Prog Biop; qys molec Biol +1987), Sun: 8 9-15°EECK80: Beak, E.

Nucl Ac1d Res (1980), 31(13) 3011-3024 ゜BENS84・ BENS88・ BENZ88a: Benz, R, and K Bauer.

Eur J Biochem (1988), Y Todoroki: 1-19° BENZ88b : Benz, R.

Ann Rev Microbiol (1988), Arc: 359-93°B ERG88: BETrg8: B) IAT86: BODE89: CAJjM90: CARU85・ Camthers, M■.

5science (1985), ;i, lQ: 281-285° CARU87 : CA-cho R87 Catron, 181. and CA Schnaitman.

J Bacteriol (1987), 1%: 4327-34゜Gene ( 198g), 2Q(1) 181-9゜CHOU74: G! 0W87: CLEMl! 1: CLIC88: CLtJN84 CRE [84: CREI88: RUZ85 CRUZ89: CWIR90: DALL90: Dallas, W.S.

J Bacteriol (1990), 1:n(9)5490-93DEGE 84・ DELA88: DEVL90: DLIΣ7: Dill, K.A.

Protein Engineering (1987), l:369-37 1゜DUCH88: DULB86: DWAR89・ E[5E85: ELLE88: EV, ANllt8: FAMEΣ9: F mouth U 2 uni FerenC1+”+ Microbiol (Inst Pa5teur) (1982), D167-169°FERE82b: FERJE83 E-Japanese-U 4゛ Ferenci, T.

Trends in Biological 5science (1984) Vol.? :44-48°FRAN87: FRB'U 89 87 yen to G: GAUS87: GET'288: GIBS88: GRAY83: GRAY84: GUAR89: GTJDM89: GUSS88: GU7M89・ GUZM90: HARD90: HASH85: HATA90: HEDE89: HHN87: H mouth N88- Heine, HG, G Francis, KS Lee, and T Ferenci.

J Bacteriol (, pri1 198g), J3:i:1730- 8゜…DA90 Ho mouth 85: HOLA89a: HOLA89b: HORV89: HOUG84゜ rrOK79: JANA89: q85° to J , JEMN89: 几IDD85: Judd, R.C.

LnfeCt Immun (1985), J4(2) 452-7゜UDD8 6 KATZ86: KATZ90: KISH85, KOBA89: KUBO89: KUHN8511: KUHN85b: KUPE90: LAM91: LUND86: MANI 1112: MANO86: MARK91: Markland er al.

Gene (1991) 109:13-19゜MCKEs5: McKern, Nhf, IJ OoDonnell, DJ Stewan , and BL C1ark.

I Gen Microbiol (1985), l;■ (Pt 1) 1- 6゜MISR88a: Misra, R, and SA Ben5on.

J Bacteriol (198g), 12Q (8) 3611-7゜N SR 8111b Misra, R, and SA Ben5on.

J Bacteriol (198g), 12Q:528-33゜MOR587 : MOR588: Morse, S.A., C-Y Chen, A. LeFaou, and T. A. Meitzner.

Rev Infect Dis (1988), IQ (Suppl 2) 530 6-10゜NICH88: N1cholson, H, WJ Becktel, and BW MAnl leWS.

Nature (1988), 115:651-56゜NICH88: N1kaido, H, and HCP Wu.

Proc Naguchi Acad Sci USA (1984), 1:1048- 52°Nl5H82: N15hiuchi, Y, and S Sakakibam.

FEBS Shie (1982), En: 2ω-2°Nl5H86: OK entry M90: ABO36 PAG feather 8: PANT90: PARD89: PARM88: ABO18: PRAS88: Pease, JHB, and DB Wemmer.

Biochem (1988), 27:8491-99゜PEAs90 Peise, JHB, RW S [orrs, and DB Wemmer .

1) roe Natl Acad Sci USA (1990), fd:5 643A1゜PE艮R84: PaR86: POTEg3: RASC86: Raxha: I, I, arid E O anti-mr.

Microbiol Rev (1986), $:401-427゜RASH 84: RHD 88a: Re1dhaar-OLson, JP, and RT 5auer.

5science (1988), 2iL:53-51°RE[D88b・ RICH86: Richards, J.H.

Nature (1986), 321:187゜RrVI87b: ROB wings 6 Roberts, S, ajId ARReesProtein signature= ring (1986), 1:59-65゜RO5Slltl: S U88: 5AUE86: Biochem (1986), 2:L:5992-98゜SG(A78) : 5CHN86: 5CHU79: 5COT87a: 500 pieces 0: S EKI 85: S HIM 11!7 FEBS Lett (1987), Praise: 165-170゜5LH77: SMrrllt5: Smjth GP.

5science (1985), 21.1315-1317゜5ODE85: SON Ei 5゜ 5TAD89: STE [85: S+einer.

BioScience Rxpu, (1985), C: 973ff.

SUMM91: 5TJNX87: TAICA85: AKE90 TAMK71: THOM85a: THOM85b Ding HOR88: 0MM82 TRIA 88: V, AM) 86: VAND90: VER586a: Vai 86 b: VOGEI jealous: “W Kingfu S78: WaL86: W month Aλ86: LK84 Wukinson, AJ, ARFersht, DM Blow, P Caner, and G. Wmbr.

Nature (1984), IQ2:187-188゜WOOD90: Procedural amendment (c) September 29, 1994

Claims (21)

[Claims] 1. A specialized target consisting of providing a population of genetic packages Developing novel binding proteins with desirable binding activity for target substances. in the step of assembling the chimera, in which each Expose one or more of the special possible bindings to where the cluster is not naturally associated with the outer surface of the package; The binding domain is fully exposed, and the plurality of different possible binding domains are fully exposed. The differentiation within is not at all amino acid positions, but at each of the changed positions. one belonging to a predetermined set of two or more amino acids one or more of the parent binding domains from which amino acids are randomly obtained. generated through at least partial random changes in defined amino acid positions. The amino acids of the set are added to the positive position in a predetermined and expected proportion. occurs, the package comes into contact with the target material, and the target material The packages are separated according to their affinity for target substances. Improvements include miniprotein sequences of fewer than 40 amino acids and at least the first at least one intrachain sharing between an amino acid position and a second amino acid position essentially encompassing each of said potential binding domains having specific cross-links. and the amino acids in the first and second positions vary depending on the population. is invariant among all of the expressed chimeric proteins and remains unchanged throughout the population. has a residual group that participates in the formation of one covalent cross-link, and the cross-link is Provided that it is in the form of a disulfide bond, the potential binding domain is 4 Micropalidic sequences with fewer than 0 amino acids. 2. In the method of claim 1, the crosslink is one disulfide bond. And the amino acids at the first and second amino acid positions are cysteine. 3. In the method of claim 2, the microprotein domain is It has a ruphide bond and the span of the bond is less than nine amino acid residues. It is. 4. In the method of claim 2, the microprotein domain is A superquadratic system with a ruphide bond and a single hairpin overall under affinity separation conditions. It bridges the sequence of amino acids whose structure is assumed. 5. In the method of claim 4, the secondary structure of the hairpin is a group consisting of the following: selected from the loop. That is, (a) one alpha helix, turn, (b) one alpha helical turn, and alpha a helix, and (c) one beta strand, turn, and betas. Tolland. 6. In the method of claim 2, the microprotein domain has two intrachain discontinuities. consisting of a ruphide bond and preferably containing two cysteine clusters. There is. 7. In the method of claim 6, the microprotein domain is 1-3, 2-4. It has a bonding pattern of two disulfide bonds. 8. In the method of claim 2, the microprotein domain comprises three intrachain discontinuities. consisting of a ruphide bond and preferably containing two cysteine clusters. There is. 9. In the method of claim 8, the microprotein domain is 1-4, 2-5. , has three disulfide bonds with a 3-6 connection pattern. 10. In the method of claim 7, the microprotein domain is one alpha There is substantial correspondence in sequence to the aconotoxin. 11. In the method of claim 9, the microprotein domain is or substantially corresponds to the sequence for omega-conotoxin. 12. In the method of claim 6, the microprotein domain is Escherichiae. Escherichia coli heat stable toxin I (STA), honey bee Toxic apamin or pumpkin seed trypsin inhibitor, scorpion toxin, caribou Dotoxin (charybdotoxin) and secretory leukocyte protease inhibitor The sequence for one microprotein selected from a group consisting of harmful substances It corresponds qualitatively. 13. In the method of claim 1, the shared cross-link is made of zinc, iron, copper, or contains metal atoms such as cobalt. 14. All methods of claims 1 to 13 provide for At least one variable amino acid position is NNT, NNG, RNG, RMG, VN Simply modified codes selected from the group consisting of T, RRS, and SNT coded by 15. All methods of claims 1 to 13 provide that the variable in the possible binding domain All of the amino acid positions belong to the group consisting of NNN, NNK, and NNS. was not coded by a simply varied cordon selected from among them. 16. All methods of claims 1 to 13 provide for Coded by a codon with complex changes in at least one variable amino acid position It was done. 17. All methods of claims 1 to 16 provide that the genetic package that can be replicated is a phage, preferably one DNA phage other than phage lambda , more preferably filamentous phages. 18. The method of claim 17, wherein the possible binding domain is a filamentous phage or an assembled The main coat protein of the fragment obtained by or fused with gene III protein in fragments that can be assembled. 19. In all the methods of claims 1 to 16, the replicable genetic package is E. scherichia coil, Salmonella typhimuri um, Pseudomonas aeruginosa, Klebsiella pneumonia, Neisseria gonorrhoeae, or Bacterial cells such as Bacillus subtilis, and the DNA The assembly further encompasses one periplasmic secretion signal sequence, and allows The binding domains are IamB protein, OmpA, OmpC, OmpF, phos phplipase A, or pilin, or segments that can be assembled. It fuses with the outer surface proteins of the bacteria. 20. All methods of claims 1 to 19 provide that the population comprises at least 105 different are characterized by the manifestation of possible binding domains, and therein for any of the possible binding domains encoded by at least one in the population. The probability exhibited by one package is at least 50%. Yes, more preferably at least 90%. 21. library displaying phages or cells, each outside of one chimera One or more of the special potential bonds as part of the surface protein. such that the potential binding domain binds to the phage or cell. The library has multiple different potentials without natural contact with the outer surface of the cells. The binding domain is exposed entirely, and two or more amino acid positions are exposed, rather than all amino acid positions. or one amino acid that belongs to a predetermined set of more amino acids. One of the parent binding domains is also randomly available at each variable position. or more predetermined amino acid positions at least in part. The multiple different potential binding domains that occur through random changes differentiation between, occurring at the position in a predetermined and expected proportion The set of amino acids is essentially a mini-protein sequence of no more than 60 amino acids. and at least a small amount between the first and second amino acid positions. The potential binding domain has at least one intrachain covalent cross-link. each of which is invariant among all chimeric proteins expressed by the population. Amino acids in the first and second positions, a shared clone that remains constant throughout the population. With these residual groups participating in the formation of the slink, the crosslink becomes a disulfide. Provided that it is a double bond, the potential binding domain is smaller than 40 residues. At least microproteins.